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Cellular mechanisms for the regulated degradation of aldolase B

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Cellular mechanisms for the regulated degradation of aldolase B
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CELLULAR MECHANISMS FOR
THE REGULATED DEGRADATION OF ALDOLASE B












By

Peter P. Susan


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1998















TABLE OF CONTENTS

page

A B S T R A C T ........................................................................................................... vi

CHAPTERS

1 IN T R O D U C T IO N ............................................................................................... 1


General Concepts of Protein Degradation..................................................... 1

Background for Fructose 1,6-Diphosphate Aldolase B................................. 3

Mechanisms for Degradation Aldolase B...................................................... 8


Hypothesis for Stress-Induced Degradation ofAldolase B.......................... 28

G general Strategy........................................................................................ 30

2 MATERIALS AND METHODS........................... .......................................... 33


Cell Lines and Culturing............................................................................. 33


Plasmid Vector Construction and Mutagenesis........................................... 35











Expressing Epitope-Tagged Aldolase B in Cell Lines.................................. 41

Immunofluorescence.................................................................................. 44

Antibodies.................................................................................................. 44

Viability Assays.......................................................................................... 48

Subcellular Fractionation............................................................................ 49

Enzym e Assays.......................................................................................... 52

Protein Analysis......................................................................................... 53

Stress-Induction of Protein D egradation..................................................... 55


Protein Degradation................................................................................... 57

3 UBIQUITINATION MEDIATES LYSOSOMAL PROTEOLYSIS OF
ALD OLA SE B................................................................................................ 59
Introduction............................................................................................... 59

In Vivo M ultiubiquitination of Aldolase B................................................... 60

Multiubiquitinated Aldolase B is Denatured and Enriched in Lysosomes.....68

Heat Stress-Induced Delivery of Aldolase A to Lysosomes Requires
Ubiquitination......................................................................................... 69

Heat Stress-Induced Proteolysis of Aldolase B Requires Ubiquitination...... 75










Ubiquitin-Mediated Autophagic Degradation Occurs in E36AB Cells..........93

4 TEMPERATURE MODULATES AUTOPHAGY AND CYTOSOLIC
PROTEOLYSIS OF ALDOLASE B............................................................... 101
Introdu ctions............................................................................................. 10 1

Ubiquitin-Independent Cytosolic Proteolysis of Aldolase B....................... 102

Temperature-Dependent Cytosolic Proteolysis in Fao Cells........................ 106


Starvation-Induced Autophagic Degradation of Aldolase B in Fao Cells.... 110

Temperature-Dependent Autophagy and Cytosolic Proteolysis.................. 118

A Model For the Degradation of Aldolase B.............................................. 123

5 SIGNAL-MEDIATED DEGRADATION OF ALDOLASE B........................... 127

Intro du action ............................................................................................... 12 7

Transient Expression of RABM Mutations in Putative Lysosome
T argeting Signals.................................................................................... 130

Starvation Induces Autophagic Degradation in HuH7 Cells........................ 139

Transient Expression Does Not Affect Starvation-Induced Degradation
o f R A B M ............................................................................................... 14 0
Site-Directed Mutations Did Not Affect Wildtype Activity of RABM........142

Glutamine Residue #111 is Required for Starvation-Induced Degradation
of A ldolase B ......................................................................................... 143
Glutamine #111 Specifically Mediates Starvation-Induced Degradation of
A ldo lase B ................................................................................................. 14 6
6 SUMMARY AND CONCLUSIONS................................................................. 151


iv










Introduction............................................................................................. 151

Autophagy and Ubiquitination.................................................................. 151


Clues from Temperature-Dependent Cytosolic Proteolysis and
Lysosomal Degradation......................................................................... 154
Signal-M ediated Targeting....................................................................... 157

Present and Future Contributions to the Field of Protein
Turnover............................................................................................... 153
REFERENCES.................................................................................................... 164

BIOGRAPHICAL SKETCH ................................................................................ 176















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CELLULAR MECHANISMS FOR
THE REGULATED DEGRADATION OF ALDOLASE B

By

Peter P. Susan

August 1998

Chairman: William A. Dunn, Jr.
Major Department: Anatomy and Cell Biology

Stress-induced degradation of abundant long-lived cytosolic housekeeping

proteins was examined using liver aldolase B as a model protein. Heat stress increases

ubiquitination that mediates autophagic degradation of long-lived proteins in E36

Chinese hamster cells. During starvation, major multiubiquitinated proteins (e.g., Ub68)

increased in lysosomes (rat liver and Fao hepatoma cells) and were antigenically

characterized as aldolase B-ubiquitin conjugates. Compared with controls, heat stress

increased endogenous aldolase A activity in lysosomes of E36 cells by >twofold. Heat

stress was non-permissive for ubiquitination in E36-derived ts20 mutant cells and failed

to increase aldolase activity in ts20 lysosomes. Myc-tagged aldolase B (RABM)

expressed in E36 cells underwent limited proteolytic processing in lysosomes that failed










to occur in heat stressed ts20 cells. The results suggested that during stress (starvation

or heat), aldolase A and aldolase B can undergo ubiquitin-mediated autophagic

degradation. Long-lived protein degradation was a continuous function of temperature,

indicating heat stress-induced rates were due to thermodynamic stimulation of chemical

reactivity. Lysosomal inhibitors distinguished proteolysis in lysosomes from that in

cytosol. Complete autophagic degradation to amino acids in lysosomes was highly

temperature-dependent compared to a relatively constant rate in cytosol. HA-tagged

human aldolase B (HAHAB) in Fao cells and RABM in E36 cells underwent proteolysis

in cytosol that had temperature-dependence paralleling complete degradation of proteins

in lysosomes. Lysosomal degradation was ubiquitin-dependent (blocked in heat stress

ts20 cells), but cytosolic proteolysis of RABM was not. Results suggest a possibly

shared temperature-dependent cytosolic mechanism that limits rates for partial cytosolic

proteolysis and complete lysosomal degradation of long-lived proteins. Three peptide

motifs for signal-mediated targeting to lysosomes during starvation occur in aldolase B.

These were mutated in RABM. Starvation-induced degradation of mutant and wildtype

RABM expressed in HuH7 human hepatoma cells were measured. Starvation-induced

degradation of RABM (aldolase B) specifically required a glutamine at residue #111

suggesting that the corresponding peptide motif, IKLDQ, is a targeting signal

functionally demonstrated in living cells. Evidence was provided for three previously

unknown mechanisms for stress-regulated degradation of aldolase B: (1) ubiquitin-










mediated autophagic degradation in lysosomes, (2) temperature-dependent cytosolic

proteolysis during heat stress, and (3) signal-mediated degradation during starvation.















CHAPTER 1:
INTRODUCTION

General Concepts of Protein Degradation

In cells, different proteins have different functions and occur at different levels as

needed. The functional activities and locations of proteins are regulated to integrate with

each other, maximizing survival. Proteins can be regulated by a variety of mechanisms,

but available concentration of each protein fundamentally determines maximal function

(Doherty and Mayer, 1992). Cells adapt to environmental change by altering amounts of

different proteins. Some increase, others decrease, and the rest are constant (Doherty

and Mayer, 1992). Such adaptation of different proteins requires preferential

mechanisms for regulating synthetic or degradative rates in response to environmental

change.

Under constant conditions, protein synthesis is independent (zero order

relationship) of the protein concentration, but degradation is directly proportional (first

order relationship) to protein concentration (Doherty and Mayer, 1992). Synthesis

increases protein concentration, causing degradation to increase until synthetic and

degradative rates are equal. In this way, a balance between synthesis and degradation

determines the available concentration of a protein. If environmental conditions change,

then cells can adapt protein concentrations by modulating synthesis or degradation










(Olson, et al., 1992; Mortimore, 1987). This study examines mechanisms of protein

degradation that respond to environmental changes.

Continual synthesis and degradation results in constant turnover of proteins

which can be described by either the fractional degradation rate or the half-life of the

protein. The fractional degradation rate of a protein (degradative rate constant), kd, is

defined as the fraction of the initial protein degraded in a given time. The kd is calculated

from measurements of labeled protein lost per time. Half-life, tv2, is defined as the time

for turnover of half the protein. Under equilibrium conditions, ka and ty, are constant and

directly related to each other by t./, = ln(2)/ kd, allowing calculation of t% from

experimentally determined kd (Doherty and Mayer, 1992).

Proteins are categorized as short-lived for ty < 1 h or long-lived for t% >1 h. For

short-lived proteins, protein concentrations respond more to changes in synthesis (Olsen,

et al., 1992). For long-lived proteins, protein concentrations respond more to changes in

degradation (Olsen, et al., 1992; Mortimore and Poso, 1987). Detailed reasoning for

this is described elsewhere (Doherty and Mayer, 1992). Many short-lived proteins utilize

a well-characterized mechanism for degradation by a cytosolic protease complex called

the proteasome (Rock, et al., 1994; Ciechanover and Schwartz, 1994; Hochstrasser,

1992). Relative to short-lived proteins, mechanisms for the degradation of long-lived

proteins are poorly characterized. This dissertation examines mechanisms for stress-

inducible degradation of a long-lived cytosolic enzyme, fructose 1,6-diphosphate

aldolase (see next section).










Mechanisms for degradation of proteins become enhanced during environmental

stress. Increased temperature (Bates, et al., 1982; Hough and Reichsteiner, 1984) or

nutrient deprivation (Mortimore and Poso, 1987) are known to increase the degradation

of long-lived proteins. Stress-induced degradative mechanisms are of special interest,

because they mediate regulated changes and themselves must be regulated (Mortimore,

et al., 1987; Mortimore and Poso, 1987; Olson, et al., 1990). Any mechanism that can

be triggered by environmental stress lends itself to experimental manipulation. Such a

mechanism can be modulated simply by changing experimental conditions. Furthermore,

mechanisms required for a stress-induced degradation can be shown to be specific by

lack of effect on basal mechanisms. For example, cells respond to starvation by

increasing the degradation of long-lived proteins. 3-Methyladenine is a drug that

specifically blocks the enhanced degradation without affecting basal degradation. 3-

Methyladenine is a potent inhibitor of autophagy, a mechanism for delivering proteins to

lysosomes for degradation. Such results provide evidence that autophagy plays a role in

enhanced degradation but not basal degradation of proteins.

Background for Fructose 1 .6-Diphosphate Aldolase B

In the next section, I discuss potential mechanisms for the degradation of the

liver isoform of fructose 1,6-diphosphate (FDP) aldolase, called aldolase B. Molecular

mechanisms for the degradation of aldolase B have not been examined, but preliminary

examination indicated potential roles for ubiquitination and signal-mediated delivery to

lysosomes. Before examining the degradation of aldolase B, this section serves to










familiarize the reader with aldolase B in relation to other aldolase isoforms. There are

two major classes (I and II) ofFDP aldolase that have no sequence homology

(Alefounder et al., 1989) and utilize different catalytic mechanisms (Rutter et al., 1966).

Class II aldolases are only found in microorganisms and are considered no further here,

but class I aldolases are represented in all taxons (Rutter et al., 1966). Microorganisms

variably lack class I aldolase. For example, some strains of E.coli contain class I

aldolase (Alefounder et al., 1989), whereas strain JM83 lacks class I activity (Sakakibara

et al., 1989). Protozoa and muticellular eukaryotes contain class I aldolases.

The FDP aldolase isoforms of mammals are the best characterized. Most studies

ofFDP aldolase degradation examine mammalian isoforms. Mammalian aldolase

isoforms are synthesized from three separately regulated genes coding different proteins.

Muscle, liver, and brain each express one predominant isoform designated A, B, and C,

respectively (Rutter et al., 1966). As such, liver aldolase and aldolase B are synonyms.

Likewise, aldolase A is also called muscle aldolase, and aldolase C is referred to as brain

aldolase. Most tissues, including embryonic, contain combinations of aldolase A and C,

but aldolase B appears to be exclusively expressed in liver and kidney cells (Rutter et al.,

1966).

Aldolase classes and isoforms are distinguished by clear differences in their

enzymatic properties. For example, class I aldolase like that in mammals is totally

functional in the presence of EDTA, but class II enzymes ofE. coli and other microbes

are completely inhibited. The three mammalian isoforms (A, B, and C) can be










distinguished by differences in specific activity, sensitivity to carboxypeptidase A, and

kinetics (Vx and Kin) for different substrates (Rutter et al., 1966).

Aldolases A, B, and C also are characterized by distinct native epitopes. Thus,

antibody to one poorly recognizes the others. However, an antibody against a specific

isoform for one animal will similarly recognize the same isoform of a different species, at

least amongst mammals (Penhoet and Rutter, 1975). Native and denatured epitopes of

aldolase B have also been shown to be distinct from each other (Reznick et al., 1985).

Chemical denaturation of aldolase B before immunization resulted in antibody that failed

to immunoprecipitate native enzyme activity but could precipitate degradative fragments

of aldolase B. Since aldolase has stable structure that spontaneously refolds into native

conformations, only fragments sufficiently denatured by degradation were detected by

antibody against the denatured aldolase B (Reznick et al., 1985). Anti-native aldolase B

had converse immunoreactivity. Thus, it was proposed that three-dimensional

conformation is important for antibody recognition of native surface epitopes, whereas

the native structure buries and masks denatured epitopes (Reznick et al., 1985).

The three dimensional structure of all class I aldolase isoforms is conserved from

bacteria to humans (Alefounder, et al., 1989). Secondary and tertiary structures of

aldolase are very stable. This is true at the quaternary level, too. Aldolase occurs as

very stable tetramers that do not undergo subunit exchange after synthesis (Lebherz,

1975; Lebherz, 1972). Different aldolase isoforms co-synthesized in the same cell

randomly associate into stable heterotetramers. Thus, immunoprecipitation with










antibody against one isoform specifically precipitates antigenically unrelated isoforms in

the same tetramer. However, surface charge and pI on different isoforms varies. Thus,

isozymes containing different ratios of two isoforms (e.g. A4, A3B, A2B2, AB3, or B4)

can be separated by isoelectric focusing (Lebherz, 1972; Penhoet and Rutter, 1975).

Comparison of X-ray crystallographic results shows that secondary and tertiary structure

between muscle, liver, and Drosophila aldolases are very close (Berthiaume et al., 1993).

Aldolase isoforms vary in their capacity to bind actin cytoskeletons. In the only

paper to measure cytoskeletal association of all three isoforms (A, B, and C),

investigators claimed that different purified isoforms of aldolase had tissue-specific

affinity for cytoskeletal preparations isolated from different tissues (Kusakabe et al.,

1997). After mixing crude cytoskeletons with a known amount of purified aldolase, they

pelleted the mixture and then only measured unbound activities in supernatants. They

failed to show removal of endogenous aldolase from cytoskeletal preparations, so

measurements could be contaminated and include competitive effects. However, their

results were consistent with other investigators in that relative tightness of binding to

actin cytoskeleton is greatest to least, aldolase A, aldolase B, then aldolase C (Clarke, et

al., 1982; O'Reilly and Clarke, 1993).

Four "isotype specific" sequences contain most of the variation and the carboxyl

terminus has the greatest diversity (Marchand et al., 1988; Paolella et al., 1984;

Rottmann et al., 1984). The carboxyl terminus is important in determining isoform-

specific catalytic properties (Berthiaume et al., 1993; Gamblin et al., 1991; Penhoet and









7
Rutter, 1975). Aldolase B has the lowest specific activity amongst aldolase isoforms and

is least sensitive to proteolytic alterations in this region. It can lose up to four C-terminal

residues without affect its enzymatic activity (Berthiaume et al., 1993; Horecker et al.,

1985). Even with 10 to 20 residues removed by carboxypeptidase, aldolase B retains

almost half its activity (Penhoet and Rutter, 1975). However, aldolase A absolutely

requires a carboxyl terminal tyrosine at residue 364 (Y364) for activity that is about 20

times greater than aldolase B, and when aldolase A loses its C-terminus the remaining

activity resembles that of aldolase B (Takahashi et al., 1989; Gamblin et al., 1991).

These results indicate that alterations in the carboxyl terminus of aldolase B (such as

epitope tagging) are less likely to affect its properties than other aldolase isoforms.

All fructose 1,6-diphosphate aldolase enzymes catalyze a reversible reaction

essential for glycolysis and gluconeogenesis. Aldolase B is the liver form of this enzyme

expressed to the exclusion of other forms of aldolase in normal hepatocytes (Asaka, et

al., 1983). Since liver is the only organ known to export glucose (Stein and Arias, 1976;

Stryer, 1988), aldolase B performs gluconeogenesis for the entire body. Liver also

provides amino acids during starvation and in three days can lose nearly half its weight

(and protein content), a faster loss than other tissues (Wing et al., 1991). In this regard,

aldolase B is an example of an abundant cytosolic protein that undergoes enhanced

degradation during starvation which yields amino acids for export to other organs. Liver

amino acids can also be converted to glucose or ketone bodies to provide energy sources

during starvation. Abundant long-lived liver enzymes that mediate












glycolysis/gluconeogenesis, like aldolase B and glyceraldehyde phosphate dehydrogenase

(GAPDH) are poised between two mutually exclusive functions: catalyzing carbohydrate

metabolism and providing amino acids for protein biosynthesis or energy metabolism.

Liver and kidney are the only tissues having predominant aldolase B expression

(Penhoet and Rutter, 1975). Both organs demonstrate enhanced degradation of proteins

during amino acid starvation (Olsen, et al. 1990). Liver and kidney also receive the

largest fraction of the body's basal blood flow, 27% and 22%, respectively, followed by

15% for muscle and 14% for brain (Guyton, 1979). This is consistent with involvement

of the two former organs in regulating serum components and contribution of aldolase B

to serum glucose and amino acids during starvation. Among aldolase isoforms, aldolase

B contributes a greater role in carbohydrate and protein metabolism that is not limited to

local cells and tissues, but extends to the entire body.

Mechanisms for Degradation Aldolase B

Inactivation by Limited Proteolysis

Alteration of aldolase A and B's carboxyl termini was proposed to down-regulate

activity (Pontremoli et al., 1982; Pontremoli et al., 1979). During starvation, aldolase B

activity is lost from liver faster than loss of immunoreactivity. Thus, investigators

suggested that starvation-induced inactivations precede total degradation of aldolase A

and B, providing more rapid down-regulation of activity (Pontremoli et al., 1979).

Inactivation happens by limited C-terminal cleavage that can maintain native








9
immunoreactivity and barely affect mobility on SDS-PAGE. One group of investigators

proposed phosphorylation near the C-terminus of aldolase as an inactivating mechanism,

but this was only demonstrated in vitro (Sygusch et al., 1990). More likely inactivation

occurs by limited proteolysis which would have a much more profound impact on

aldolase A activity than on aldolase B activity (previous section, last paragraph).

The best characterized mechanism for aldolase inactivation is limited proteolysis

by a dipeptidyl (two residues per cleavage) carboxypeptidase on lysosomes (Pontremoli

and Melloni, 1986; Horecker et al., 1985). The peptidase, cathepsin M, was defined as

a cathepsin B or L-like activity associated with the cytosolic surface of lysosomal

membranes. During starvation, a lysosomal matrix cathepsin B/L associates with

lysosomal membranes, acquires activity at neutral pH, and becomes exposed to the

cytosolic compartment as cathepsin M (Pontremoli et al., 1984; Pontremoli et al., 1982).

Specific cleavage sites have been characterized in vitro (Horecker et al., 1985).

Starvation-induced in vivo loss of liver aldolase specific activity correlated with

loss of carboxyl terminal tyrosine residues which was estimated by isolating aldolase B

and measuring lost tyrosine content in an acid soluble peptide released from the C-

terminus with subtilisin (Pontremoli et al., 1982). According to such experiments,

inactivated aldolase B constitutes about 40% of the aldolase in liver after 60 hours of

starvation. Most of the inactivated aldolase B must occur in cytosol, because only a

small fraction of total aldolase (about 10%) is associated with pelletable fractions from

liver (Kominami et al., 1983; Kopitz et al., 1990). Moreover, intralysosomal










degradation of aldolase is rapid (see below), precluding accumulation of an inactivated

form in such organelles. The results are consistent with inactivation occurring in the

cytosolic compartment, albeit by an activity associated with the cytosolic surface of

lysosomes.

In Vitro Denaturation of Aldolase and Need for In Vivo Mechanism

Except for 20 "loose" amino acid residues at the carboxyl terminus, the stability

of aldolase structure resists proteolysis and requires denaturation for rapid in vitro

proteolysis to proceed. In optimized conditions with cathepsin D, only about 20 amino

acids of aldolase A can be digested from its carboxyl terminus (Offermann et al., 1983).

In vitro proteolysis with either meprin (a metalloproteinase) or a mixture of lysosomal

proteases produces only a slight increase in SDS-PAGE mobility, and the remaining part

of aldolase A has a thermal stability identical to the native enzyme (Bond and Offermann,

1981). Purified aldolase B digested with a lysosomal extract also only undergoes

limited proteolysis, losing some but not all its activity (Chappel et al., 1978). However,

denaturing pretreatment with disulfides like glutathione (Offermann et al., 1983) or

cystine (Bond and Offermann, 1981) permits extensive proteolysis to occur. Given

this, there must be a denaturingg" mechanism in vivo to allow degradative turnover of

aldolase to occur. Interestingly, aldolase B sequestered in vivo and isolated with

lysosomes is susceptible to more extensive in vitro proteolysis in the lysosomal

preparations (Kominami, et al., 1983; Ueno and Kominami, 1991). Apparently,










aldolase B becomes sensitized to proteolysis by a mechanism in cytosol before

sequestration or in intact lysosomes after sequestration.

After loading aldolase A into endosomes at 19C, temperature can be raised to

37C allowing rapid fusion of endosomes with lysosomes. Thus, intralysosomal

degradation can be measured. By this method, native or variously denatured and

inactivated aldolases all degrade rapidly with similar rates (t, < 10 min). Since its t, is

normally many hours in cytosol, sequestration appears rate limiting for lysosomal

degradation of aldolase (Bond and Aronson, 1983). The results of the endocytic loading

experiments indicate that a mechanism for denaturing and sensitizing aldolase to

proteolytic attack can occur in lysosomes or other organelles of the endosomal pathway.

Thus, a cytosolic denaturing mechanism is not necessary for intralysosomal degradation

of aldolase, but a role in delivery of aldolase to lysosomes cannot be excluded.

The tetramrneric structure of aldolase is well established (Lebherz, 1972). This

quaterenary structure seems important for aldolase stability. Recently, Beemrnink and

Tolan have identified specific amino acids that mediate subunit interaction between

aldolase monomers (Beemrnink and Tolan, 1996). Significantly, a mutant with only two

amino acid changes retains enzymatic activity but exists as monomers. These monomers

(and dimers created with single amino acid mutations) are more sensitive to chemical or

thermal inactivation, indicating "looser" structure. Thus, tetrameric association

improves structural stability.










Lysosomes are acidic inside (pH -5), and reversible in vitro dissociation of

aldolase into monomers occurs at pH < 6.0 (Beernink and Tolan, 1996). Acidic pH

affecting adolase structure is also indicated by reduced enzymatic activity. Thus,

intralysosomal pH would have a denaturing effect that could permit lysosomal

proteolysis. However, other investigators incubated aldolase B with crude lysosomal

hydrolases at acidic pH and failed to get significant proteolysis (Chappel et al., 1978).

Apparently, low pH is insufficient to permit further proteolytic attack, and aldolase

denaturation must require other factors. Consistent with this, lysosomes purified from

liver contain detectable aldolase B which is susceptible to proteolysis when the intact

lysosomes are incubated in vitro at pH < 5.5 (Kominami, et al., 1983; Ueno and

Kominami, 1991). The endocytic loading experiments described above indicate

lysosomes (or an endocytic compartment) must contain denaturing factors, but this does

not exclude the possibility of a cytosolic denaturation of aldolase B before delivery to

lysosomes.

Aldolase A has been radiolabeled, inactivated and denatured, then microinjected

into cultured cells (Hopgood et al., 1988; Knowles et al., 1989). The procedure delivers

the enzyme into cytosol where it normally resides. As with endocytic loading,

degradation rates for aldolase were similar whether the enzyme was native, inactivated,

or denatured. Denaturation of aldolase is not rate limiting for degradative steps before

lysosomes as well as within them. Degradation of aldolase microinjected into cytosol

matched expected turnover for aldolase (ty = 30 hours) which was much slower than for










aldolase loaded into lysosomes (ty, < 10 minutes). Assuming that degradation occurs

within lysosomes, this suggest that sequestration of aldolase is rate-limiting for its

turnover (Bond and Aronson, 1983; Bond and Offermann, 1981; Hopgood et al., 1988;

Knowles et al., 1989).

Though in vitro studies indicate denaturation of aldolase structure is necessary

for proteolysis, in vivo denaturation is not rate-limiting for delivery to or degradation

within lysosomes. These data support a model in which aldolase delivery to lysosomes is

rate limiting followed by rapid intralysosomal proteolysis which would need a faster

denaturing mechanism. Lysosomal acidity might facilitate denaturation of aldolase, but

acidity alone is insufficient for sensitizing stable aldolase structure to attack by acid

hydrolases. The above data do not exclude a cytosolic denaturing mechanism for

aldolase, but indicate that such a mechanism is not rate limiting and not necessary for

intralysosomal proteolysis. The next two sections review mechanisms for the delivery of

cytosolic proteins to the lysosomal lumen, a process that appears rate-limiting for

aldolase degradation.

Autophagy

Autophagy is the sequestration of cytoplasm into vesicles for intralysosomal

degradation and is the only mechanism proposed for the complete degradative turnover

of aldolase. There are two forms of autophagy: macroautophagy and microautophagy.

Commonly, investigators use the term "autophagy" to mean macroautophagy which is










the better characterized form. Likewise, "autophagy" used here refers to

macroautophagy, and reference to "microautophagy" will be explicit.

Autophagy (macroautophagy) begins with a ribosome-free portion of

endoplasmic reticulum engulfing a portion of cytoplasm. Autophagy non-selectively

sequesters cytosol and organelles into distinct autophagic vacuoles. The autophagic

vacuoles mature including a process of acidification. Finally, mature autophagic

vacuoles fuse with lysosomes producing autolysosomes in which degradation occurs

(Dunn, 1990; Dunn, 1990). Enhanced autophagy is initiated by amino acid starvation

and is also regulated by hormones (Hendil et al., 1990; Seglen and Bohley, 1992). In the

model of Figure 1-1, non-selective autophagy is represented by the upper pathway in the

diagram. The lower pathway of Figure 1-1 (Receptor-Mediated Targeting) is discussed

in the next section.

Microautophagy seems simpler than macroautophagy. During microautophagy

the lysosomal membrane itself invaginates, extending a finger of cytosol into the

lysosome. This protrusion pinches off producing an intralysosomal vesicle that gets

degraded with its cytosolic content. Apparently, microautophagy can occur in vitro, but

the complexity of macroautophagy has not been reconstitiuted (Seglen and Bohley,

1992). Unlike macroautophagy which has discrete autophagic vacuoles, microautophagy

fails to produce separable organellar compartments. Thus, microautophagy requires

time-consuming electron microscopy to demonstrate its existence and remains poorly

characterized (Seglen and Bohley, 1992).

































Figure 1-1: Mechanisms for Stress-Induced Degradation of Cytosolic Proteins in
Lysosomes. Autophagy (upper pathway) and receptor-mediated targeting (lower
pathway) were proposed for stress-induced delivery ofcytosolic proteins to lysosomes
for degradation; the arbitrary cytosolic protein is shown as a tetramer (aldolase B occurs
as a tetramer); components of the pathways are labeled on the diagram; processes are
labeled by boxed numbers: 1, association with or engulfminent by autophagic membranes;
2, sequestration into double-membrane bound autophagic vacuole; 3, maturation of
autophagic vacuole (acidification and acquisition of lysosomal hydrolases); 4, proteolysis
into polypeptide fragments; 5, complete degradation to amino acids; 6, disassembly and
denaturation of structure by an unknown factor; 7, association with a receptor complex
on the lysosomal surface; 8, translocation across the lysosomal membrane.



Selective mechanisms of autophagy exist. Methylotrophic yeast use a selective

mechanism of autophagy to degrade peroxisomes when switched from methanol to a

different carbon source, and electron microscopic morphology shows a mechanism









16
topologically identical to microautophagy (Tuttle et al., 1993). Occurrence of selective

microautophagy in higher organisms has not been demonstrated, and a role for

microautophagy in degradation of aldolase has not been studied. If selective autophagy

does occur for aldolase, then a receptor-mediated complex would be required for

selectivity. Such a receptor complex could form on the lysosomal surface (Fig. 1-1,

lower pathway), followed by microautophagic sequestration. However, receptor

function does not distinguish microautophagy and macroautophagy, so Figure 1-1 only

distinguishes non-selective autophagy (upper pathway) from a hypothetical selective

process that might include microautophagy (lower pathway).

Autophagy (macroautophagy) is a subject of active research, producing almost

300 related papers in just the last five years. FDP aldolases are generally abundant,

commonly known, cytosolic enzyme, and the muscle isoform, aldolase A, is

commercially available. Aldolase A and aldolase B have been used as markers for

autophagic uptake of cytosol into lysosomes, and degradation of aldolase by autophagy

is well established (Henell et al., 1987; Kominami et al., 1983; Kopitz et al., 1990;

Seglen and Gordon, 1982; Ueno et al., 1990). Per 0. Seglen's laboratory briefly treated

starved hepatocytes with cycloheximide to prevent new protein synthesis and estimated

degradation rates by loss of enzyme activity. Incubations were short to avoid depletion

of autophagic factors (continual autophagy requires new protein synthesis). A lysosomal

inhibitor (leupeptin) was used to estimate how much degradation occurred in lysosomes.

In this way, starvation-induced degradation of aldolase B was lysosomal occurring at










3.60.1 %/h. Other cytosolic enzymes with widely different half-lives were similarly

tested. As expected, they had very different total degradation rates. However,

lysosomal degradation (3.3-5.3 %/h) and rates of accumulation in organelles during

lysosomal inhibition (3.1-3.7 %/h) were similar for all the enzymes. These rates match

rates of starvation-induced autophagy (3-5 %/h) and were blocked with 3-methyladenine

a specific inhibitor of autophagy. Thus, Seglen concluded that cytosolic enzymes,

including aldolase B, are degraded via non-specific autophagy (Kopitz et al., 1990).

Interestingly, of all the enzymes tested by Seglen, only two were exclusively degraded in

lysosomes, aldolase B and lactate dehydrogenase H (Kopitz et al., 1990).

Coincidentally, both aldolase B and lactate dehydrogenase H contain sequence motifs for

receptor-mediated targeting to lysosomes for degradation.

Receptor Mediated Targeting to Lysosomes

A pentapeptide sequence (KFERQ) of RNAse A was shown to mediate its

delivery to lysosomes for degradation during nutrient deprivation (Dice and Chiang,

1988). Characterization of this signal identified a motif contained in a subset of cellular

proteins that undergo enhanced degradation in lysosomes during nutrient deprivation

(Wing et al., 1991). The motif has been proposed as a binding site for a molecular

chaperone called HSC73 which then delivers motif-containing proteins into lysosomes in

an ATP-dependent manner (Chiang et al., 1989). The mechanism (Figure 1-1, lower

pathway) also requires intralysosomal HSP70, and a recently identified lysosomal

membrane receptor, LGP96 (Cuervo, et al. 1996). J. Fred Dice proposed that this










pathway occurs by a mechanism analogous to transmembrane transport of proteins

during organellar biogenesis (Dice and Chiang, 1988; Terlecky et al., 1992; Wing et al.,

1991).

Recently, Dice's group made a major advance by identifying a receptor protein in

the target membrane of lysosomes that mediated transmembrane translocation (Cuervo

and Dice, 1996). The lysosomal membrane protein, LGP96, was demonstrated to be a

rate limiting component in this degradative pathway. CHO cells overexpressing human

LGP96 by two to three fold had correspondingly increased degradation of long-lived

proteins. Furthermore, lysosomes isolated from these cells had two to three fold higher

ATP-dependent uptake of the glycolytic enzyme GAPDH (glyceraldehyde 3-phosphate

dehydrogenase), a known substrate for his pathway (Cuervo and Dice, 1996).

Unfortunately, GAPDH does not contain sequence matching previously established

criteria for the receptor-mediated targeting motif The previous criteria define necessity

for an "essential" glutamine, but in GAPDH, asparagine apparently can substitute for

glutamine (Dice, personal communication). As just described, receptor function has been

demonstrated in living cells; however, evidence for in vivo function of the signal peptide

is lacking.

A recent study found that the conformation of signal motifs were inappropriate to

mediate the receptor-mediated lysosomal targeting pathway (Gorinsky et al., 1996).

Some proteins known to contain motifs for the pathway, including RNAse A, also have

known three dimensional structures. The peptide signal motifs are supposed to be










recognized by cytosolic HSC73 which is required for delivery to lysosomes. However,

signals on proteins of known structure are either embedded or occurred in a-helical

conformations. Since hsp70-type chaperones require extended conformations for

recognition, the investigators concluded that HSC73 would required other unknown

factors to relax a substrate protein's structure and allow signal-mediated targeting to

lysosomes to occur (Gorinsky et al., 1996). The lower pathway in Figure 1-

1 summarizes receptor-mediated targeting to lysosomes, including an unknown factor

that alters the structure of the cytosolic protein.

Except for the work presented in this dissertation, no studies have examined any

aldolase isoform as a substrate for the receptor-mediated pathway. Aldolase A binds

very well to GAPDH presumably for greater glycolytic efficiency (Verlessy and Vas,

1992). Both these proteins appear to be regulated at similar high concentrations in cells

(Verlessy and Vas, 1992), and aldolase B and GAPDH undergo similar starvation-

induced degradation in cultured cells (Kopitz, et al., 1990). Does aldolase B follow

receptor-mediated targeting to lysosomes like GAPDH?

All vertebrate aldolases contain a conserved motif (Fig. 1-2, residues 12-16) for

receptor-mediated targeting of cytosolic proteins into lysosomes (Dice and Chiang,

1988; Zhang et al., 1995). In mammalian aldolase B, two additional sequences for the

lysosomal targeting motif were found (Fig. 1-2, residues 58-62 and 107-111). Whether

any of these three motifs are functional was unknown. Though the lysosomal targeting

motif has rather broad criteria (Dice and Chiang, 1988), an aldolase-sized (40 kD)














MAHRFPALT S EQKKELSEI AQRIVANGKGILAADESVGTMGNR
MAHRFPALTQEQKKELSEI AQRIVANGKGILAADESVGTMGNR
18

LQRIKVENTEENRRBFRELLFSVDNSISQSIGGVILFHETLYQKDS
LQRIKVENTEENRRQFRE I LFSVDNSISQSIGGVILFHETLYQKDS
44 Qlll

QGKLFRNILKEKGIVVGIKLD GGAPLAGTNKETrIQGLDGLSER
QGKLFRNILKEKGIVVGIKLDOGGAPLAGTNKETTIQGLDGLSER
90

CAQYKKDGVDFGKWRAVLRIS DQCPSSLAIQENANALARYASIC
CAQYKKDGVDFGKWRAVLRIADQCPSSLAIQENANALARYASIC

135QQNGLVPIVEPEVLPDGDHDLEHCQYVSEKVLAAVYKALNDHH


QQNGLVPIVEPEV I PDGDHDLEHCQYVT SEKVLAAVYKALNDHH
QQNGLVPIVEPEV I PDGDI-DLEHCQYVTEKVLAAVYKALNDHH
179
VYLEGTLLKPNMLTAGHACTKKYTPEQVAMATVTALHRTVPAA
VYLEGTLLKPNMVTAGHACTKKYTPEQVAMATVTALHRTVPAA
221
VP S ICFLSGGMSEEDATLNLNAIYRCPLPRPWKLSFSYGR ALQAS
VPGICFLSGGMSEEDATLNLNAINLCPLPKPWKLSFSYGKALQAS
265

ALAAWGGKAANKKATQEAFMKRAV ANCQGQYVHTGSSGAAS
ALAAWGGKAANKEATQEAFMKRAMANCKGQYVHTGSSGAAS
300

TQSLFTA SYTY
TQSLFTACYTY
354 364

Figure 1-2: Amino Acid Sequences ofAldolase B Isoforms Used in This Study. The
upper and lower sequences are for rat and human liver aldolase, respectively. Boldface
indicates non-identical residues. Underline indicates pentapeptide signal motifs for
receptor-mediated lysosomal targeting. Large arrows point to essential glutamines of the
signals as indicated (sequence data from Tsutsumi et al., 1984 and Paolella et al., 1984).









protein would have only a 7% chance of randomly containing three such signal motifs.

In addition to the motifs, aldolase B has properties similar to other substrates of this

signal-mediated degradative mechanism: (1) long-lived, (2) cytosolic, (3) housekeeping

protein, and (4) degraded in lysosomes by enhanced proteolysis during nutrient

withdrawal. Furthermore, aldolase is closely associated with another glycolytic enzyme

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is an established substrate

for receptor-mediated targeting to lysosomes (Aniento et al., 1993). Aldolase and

GAPDH form a complex that facilitates their sequential roles in glycolysis (Verlessy and

Vas, 1992), both are very abundant, and their in vivo turnover rates are very similar

(Kuehl and Sumsion, 1970), suggesting that they could share degradative mechanisms.

Furthermore, the receptor-mediated pathway proceeds by transmembrane translocation

into lysosomes by a mechanism like that oforganellar biogenesis. Coincidentally, the

aldolase isoform of Trypanosoma brucei (45% identical to aldolase B) undergoes

transmembrane transport during biogenesis of the unique glycolytic organelle

of this protozoan (Marchand et al., 1988). Together, these facts suggested that aldolase

B was a likely candidate for receptor-mediated targeting to lysosomes via the proposed

transmembrane transport mechanism.

Ubiquitination and the Degradation of Long-lived Proteins

Ubiquitination is an orderly process whereby a 76-amino acid polypeptide,

ubiquitin, is covalently conjugated to other proteins at its carboxyl terminus. In a series

of transfers, three enzymes (El, E2, and E3) covalently bind and pass ubiquitin to the









22
next protein. El called ubiquitin-activating enzyme, first conjugates ubiquitin's carboxyl

terminus. This step is obligatory, and cell lines with temperature-sensitive ubiquitination

have defects traced to mutations in El (Kulka, et al., 1988; Chowdary, et al., 1994). El

transfers ubiquitin to an E2 which transfers it to an E3 which finally conjugates the

ubiquitin to a target protein (sometimes, the E3 step is skipped). Most protein

ubiquitination requires a single El protein, but E2 and E3 enzymes occur as families that

regulate and confer specificity for ubiquitination. This arrangement explains why genetic

defects in general ubiquitination only occur in El enzymes (Ciechanover and Schwartz,

1994; Hochstrasser, 1992).

Cells die without ubiquitination. The process has been implicated in a wide

variety of cell functions reviewed elsewhere (Hochstrasser, 1996; Jentsch, 1992).

Ubiquitination was originally discovered in the rapid degradation of short-lived and

abnormal proteins by cytosolic proteases, and this role remains the best characterized

(Hershko and Ciechanover, 1992). Heat stress causes enhanced degradation of long-

lived proteins in E36 Chinese hamster lung cells. This heat-stress induced degradation

occurs in lysosomes via autophagy and requires ubiquitin-activating enzyme El (Gropper

et al., 1991; Handley-Gearhart et al., 1994). However, specific long-lived proteins that

utilize this ubiquitin-mediated autophagic mechanism have not been identified.

Whether for short-lived or long-lived proteins, ubiquitin-mediated turnover

involves attachment of multiple ubiquitins on a protein targeted for degradation

(Hershko and Ciechanover, 1992). A ubiquitinated protein (a.k.a. ubiquitin conjugate) is









23
a substrate for further ubiquitination, and additional ubiquitins preferentially conjugate to

already attached ubiquitin. A chain of ubiquitins is built on a protein to be targeted. The

multiubiquitin chain then acts as a signal for degradation of the targeted protein. Each

ubiquitin adds an additional 76 amino acids to the protein, and successive intermediates

of multiubiquitination can be demonstrated as a ladder of bands on SDS-PAGE that

contain both ubiquitin and the targeted protein (Chau et al., 1989; Hershko and

Ciechanover, 1992). Multiubiquitination is a well-established signal for stress-induced

degradation of short-lived proteins by a major protease complex in cytosol. Though

total long-lived proteins undergo ubiquitin-mediated stress-induced degradation in

lysosomes, a role for multiubiquitination has not been established for lysosomal

degradation of any specific cytosolic protein.

Recently, work in the laboratory of William A. Dunn, Jr. demonstrated a

connection between ubiquitin and the long-lived protein aldolase B. The evidence

includes data presented in this dissertation, two meeting abstracts, and a manuscript

which has been submitted (Lenk et al., Submitted 1998; Susan and Dunn, 1996; Susan et

al., 1995). Together the data support that aldolase B is multiubiquitinated in vivo and

suggest that ubiquitination is involved with stress-induced autophagic degradation of

aldolase B in lysosomes.

S. E. Lenk and William A. Dunn, Jr. provided the first evidence that aldolase B

has ubiquitinated forms (Figs. 1-3 and 1-4), including a major 68 kD form (Ub68)

enriched in lysosomes during nutrient deprivation (Lenk et al., Submitted 1998; Susan et
























Figure 1-3: Characterization of A Major Ubiquitin-Protein Conjugate Enriched in
Autophagic Vacuoles. a) Rats were starved to induce autophagy and lysosomal uptake
of ubiquitinated proteins. Subcellular fractions of liver were prepared, and equal protein
from cytosolic (Cy), lysosome-enriched (Ly), and autophagic vacuole-enriched (AV)
fractions were run on SDS-PAGE, western blotted, and labeled with antibody against a
major ubiquitinated protein (anti-Ub68). Note major bands at 68 kD and cross-reactivity
to a 40 kD protein in cytosol. The 40 kD protein was identified as aldolase B by peptide
sequence analysis, b) Cytosol was circulated on an anti-Ub68 column, washed, eluted,
and preparative SDS-PAGE performed. A gel strip was stained with Coomassie blue R-
250 (CB), and the remaining gel was blotted and cut into strips individually stained with
anti-Ub68 (Ub68) or anti-ubiquitin (Ub). Ub68 and aldolase are indicated at 68 kD and
40 kD, respectively. Arrowheads indicate positions of bands suggestive of intermediates
of multiubiquitination of aldolase (from Lenk, et al., Submitted 1998).







a. Cy Ly AV b.
r.v




68 100> -, *- Ub6i

40
40 D,'Wu


Aldolase


Ub68 Ub


I|


-9
^f


F


anti-Ub68


3 sommii^^^ ^^^^


























Figure 1-4: Amino Acid Starvation Increases Lysosomal Association of Putative
Ubiquitinated Aldolase B via Autophagy. Fao rat hepatoma cells were incubated on
media with or without amino acids (AA) and the autophagy inhibitor 3-methyladenine
(3MA) as indicated. Sub-cellular fractions were collected, and equal protein of
lysosome-enriched fractions were run on SDS-PAGE, western blotted, and stained with
antibodies against Ub68 and ubiquitin (Ub). Positions of molecular weight standards and
Ub68 (arrowhead) are indicated (from Lenk et al., Submitted 1998).










Anti-Ub68


205-



116-

97-



66-


45-


29-


AA
3MA


+


Anti-Ub









28
al., 1995). Aldolase A and B are long-lived proteins known to undergo degradation by

autophagy (Henell et al., 1987; Kominami et al., 1983; Kuehl and Sumsion, 1970; Ueno

and Kominami, 1991; Ueno et al., 1990). It has been determined that amino acid

deprivation (starvation) rapidly enhances autophagy in cultured cells (Kopitz et al., 1990;

Seglen and Gordon, 1982; Ueno et al., 1990). Since Ub68 increases in lysosomes under

similar conditions (Fig. 1-4), the evidence suggested that ubiquitination might play a role

in stress-induced autophagic degradation of aldolase B.

Hypothesis for Stress-Induced Degradation of Aldolase B

The field of protein degradation has made great progress in determining

molecular mechanisms for the degradation of short-lived proteins via a cytosolic protease

complex (the proteasome); however, long-lived proteins which are generally thought to

be degraded in lysosomes have relatively poorly characterized degradative mechanisms.

Cellular degradative mechanisms that respond to environmental changes facilitate

experimental characterization. Starvation (amino acid and serum deprivation) and heat

stress can induce regulated mechanisms for the degradation of long-lived proteins.

Figure 1-1 presents two pathways proposed for the stress-induced delivery of cytosolic

proteins to lysosomes for degradation: autophagy or receptor-mediated targeting (lower

pathway).

In the simplified diagram of Figure 1-1, only topological changes in autophagy

(upper pathway) are shown for a tetrameric cytosolic protein sequestered into an

autophagic vacuole (steps 1 and 2) that fuses with lysosomes (step 3). Heat stress









29
induces autophagic degradation that requires ubiquitination, but specific proteins that are

ubiquitinated during stress-induced autophagy have not been identified. Aldolase B is

known to undergo autophagy, and a putative ubiquitinated form aldolase B associates

with autophagic vacuoles and lysosomes during starvation. To establish a specific

protein for ubiquitin-mediated autophagy, we examined aldolase B as a likely substrate

for ubiquitin-mediated autophagy.

In Figure 1-1, receptor-mediated targeting to lysosomes is also drawn showing

required components (lower pathway). Since established substrates for this pathway

have conformations that would prevent receptor recognition, unknown factors (smallest

circles) have been proposed to relax the structure of substrate proteins (first arrow)

which probably includes disassembly of subunits from quaternary structures (rough-

drawn oval with small circles attached). An exposed signal then mediates assembly of a

complex on the lysosomal surface (second arrow), including the HSC73 chaperone

(medium gray square), the substrate protein (extended coils), the lysosomal membrane

protein LGP96 (darkest rectangle), and possibly other factors (small circles).

Transmembrane translocation (third arrow) also requires an intralysosomal HSP70

chaperone (dark gray square). Aldolase B has characteristics similar to known substrates

for receptor-mediated targeting to lysosomes, but this mechanism was not examined for

any aldolase. Evidence will be shown that ubiquitinated forms of aldolase B have a more

denatured conformation. If aldolase B follows receptor-mediated degradation, then

ubiquitin could represent the unknown factor needed to relax substrate structure.










The relationship between receptor-mediated targeting of cytosolic proteins to

lysosomes and ubiquitin-mediated autophagic degradation had not been examined. Since

aldolase B was a potential substrate for both pathways, I hypothesized that during stress,

aldolase B requires both ubiquitination and a receptor-mediated targeting signal for

enhanced degradation in lysosomes.

General Strategy

I adopted the hypothesis that during stress, aldolase B requires both

ubiquitination and a receptor-mediated targeting signal for enhanced degradation. The

two requirements in this hypothesis were separately tested: ubiquitination and a receptor-

mediated targeting signal. In this regard, there were two corresponding aims of this

investigation: Aim #1, perturb ubiquitination and examine the effects on stress-induced

delivery of aldolase B to lysosomes; Aim #2, mutate potential lysosomal targeting signals

and examine effects on starvation-induced degradation of aldolase B.

My first aim was to determine whether stress-induced degradation of aldolase B

requires ubiquitination. Antibodies were raised against aldolase B expressed in and

isolated from E. coli. Since bacteria lack ubiquitin, the antibodies were produced against

antigen that did not contain ubiquitin or ubiquitin-conjugated proteins. With the

antibodies, the presence of aldolase B epitopes in a major 68 kD ubiquitinated protein

(Figs. 1-3 and 1-4, Ub68) and other ubiquitin conjugates was confirmed in subcellular

fractions from rat liver. Epitope-tagged aldolase B was expressed in E36 (parent) and

ts20 (ubiquitination mutant) cells previously used to establish ubiquitin-dependency for










heat stress-induced autophagic degradation of long-lived proteins. By examining

changes in the endogenous aldolase A and exogenous aldolase B associated with

pelletable subcellular fractions, evidence was found that these aldolase isoforms require

ubiquitination for autophagic degradation in lysosomes during heat stress. This

supported my hypothesis that stress-induced degradation of aldolase B requires

ubiquitination.

An attempt was made to use protein degradation measurements to confirm that

heat stress-induced degradation of aldolase B requires ubiquitination. Degradation of

aldolase B was found to utilize a temperature-dependent cytosolic proteolytic

mechanism. The cytosolic proteolysis of aldolase B at heat stress temperatures was

similar in magnitude to induced ubiquitin-mediated autophagic degradation. Since the

cytosolic mechanism turned out to be ubiquitin-independent, degradation measurements

could not confirm ubiquitin-mediated degradation of aldolase B via lysosomes.

However, the results demonstrate that mechanisms for degradation of aldolase B include

a novel cytosolic proteolysis.

My second aim was to test whether a receptor-mediated targeting signal was

required for stress-induced degradation of aldolase B. A sequence motif has been

defined for targeting cytosolic proteins to lysosomes for degradation during nutrient

deprivation, and aldolase B contains three sequences that match the motif (Fig. 1-2).

Depriving liver-derived cell lines of serum and amino acids causes starvation-induced

degradation of long-lived proteins including aldolase B. Vectors were constructed

expressing epitope-tagged aldolase B and used site-directed mutagenesis to disrupt the









32
putative targeting signals. Wildtype and mutant aldolase B proteins were expressed and

assayed for starvation-induced degradation. Starvation causes enhanced autophagic

degradation of aldolase B expressed in cultured hepatoma cells, and this enhanced

degradation specifically required a targeting signal that includes glutamine residue #111.

This supported my hypothesis that stress-induced degradation of aldolase B utilizes a

receptor-mediated targeting signal.














CHAPTER 2:
MATERIALS AND METHODS

Cell Lines and Culturing
General Maintenance

Except for temperature (see following subsections), all cell lines were maintained

similarly using standard sterile cell culturing techniques. Except where indicated, all

supplies were obtained from Fisher Scientific, Inc. The term "standard culture

conditions" refers to maintenance in DMEM (Sigma #D-5648), 2.2 g/1 NaHCO3, and

10% FBS (Biocell #6201-00) in a 5% CO2 atmosphere, and the standard medium for

stably transfected cells included 0.3 mg/ml active G418 (GIBCO BRL #11811-031).

Cultures were fed every 3-4 days and passage before complete confluency. For

passages, cell sheets were rinsed with DPBS (Sigma #D-5652) followed by lx trypsin-

EDTA (Sigma #T4174) in DPBS for 4-8 minutes at room temperature or 37C as

needed. Passages to amplify and maintain cultures for experiments were split 1:10 to

1:50 (area:area), and very fast growing lines that tolerated thin splits were done down to

1:80. Since trypsin/EDTA diluted 1:10 or more with 10% FBS did not affect cell sheets

during 30 min. at 37C, some thin splits (at least 1:30 into medium with 10%FBS) were

directly plated without pelleting to remove trypsin. For cultures using this short cut,

attachment times, spreading times, growth rates, and experimental results were

unaltered. Passages to replenish frozen stocks were always split heavily at 1:3 to 1:6

from freshly thawed stocks grown to near confluency. For new frozen stocks, cells
33








34
were suspended in medium supplemented with 10% DMSO (Sigma #D-2650), incubated

1-2 h at minus 20C, then at minus 80C overnight, and stored at minus 80C for up to

two months or transferred to liquid nitrogen for longer storage times.

Heat Stress-Inducible E36 Cells and Ubiquitination Mutant

Alan Schwartz kindly provided cell lines: E36 (parent), ts20 (mutant with

temperature sensitive ubiquitin-activating enzyme El ), and ts20E 1 c2 (mutant rescued by

wild-type human El) Chinese hamster lung cell lines. These cells are well characterized

for thermal control of ubiquitin-activating enzyme El activity and together have

demonstrated that El-mediated ubiquitination is required for heat stress-induced

degradation of long-lived proteins ((Handley-Gearhart et al., 1994); (Handley-Gearhart

et al., 1994); (Trausch et al., 1993); (Lenk et al., 1992); (Schwartz et al., 1992);

(Gropper et al., 1991); (Kulka et al., 1988)).

Starvation-Inducible Cell Lines

William A. Dunn, Jr. provided Fao (rat hepatoma) and HuH7 (human hepatoma)

cell lines. The Fao cell line originates from a rat hepatocellular carcinoma (Reuber,

1961), and this derivation is well documented (Deschatrette and Weiss, 1974). Fao cells

retain a dozen liver-specific characteristics examined by Mary C. Weiss, including some

endogenous expression of aldolase B (Deschatrette et al., 1979; Deschatrette and Weiss,

1974).

The HuH7 cell line was isolated from a well differentiated carcinoma of a

Japanese man and shown to secrete 16 different plasma proteins associated with liver










function (Nakabayashi et al., 1982). Seven expected carbohydrate-metabolizing

activities were present in HuH7 cells, but for two of these, liver-specific isoforms,

pyruvate kinase L and a low-Km hexokinase, were not detected (Nakabayashi et al.,

1982). BHK (baby hamster kidney), and NRK (normal rat kidney) cell lines were

examined briefly during transient transfections.

Plasmid Vector Construction and Mutagenesis

General Molecular Biological Methods

Basic methods were performed essentially as described in Current Protocols in

Molecular Biology (Ausubel, et al., 1994). Except where noted, all supplies came from

Fisher Scientific, Inc. Kits for DNA preparations were from Qiagen and Promega.

Restriction digestions, ligations, other DNA modifications, and PCR utilized supplies

from Promega and New England Biolabs, except as noted below.

PCR Primers and DNA Sequencing

At the University of Florida, the DNA Synthesis Core Laboratory provided all

oligonucleotide primers that William A. Dunn, Jr. or Peter P. Susan designed for PCR.

The University of Florida DNA Sequencing Core Laboratory sequenced parts of

plasmid vectors that were altered, or we did DNA sequencing with a SequenaseTm kit

(U.S. Biochemical Corporation).

Expression Vectors for Epitope-Tagged Aldolase B

Kiichi Ishikawa provided pRAB 1710 Amp+ (Tsutsumi et al., 1984), a plasmid

containing the cDNA of rat aldolase B (RAB) used as a PCR template in a reaction

containing two primers (Fig. 2-1). PCR solutions were prepared according to










manufacturer's specifications (Promega) and then run through 40 cycles (each cycle: 1

min. at 94C, 1 min. at 52C, and 2 min. at 72C), yielding DNA coding for rat aldolase

B tagged with the 9E10 myc epitope at the carboxyl terminus (RABM). After

restriction, the product was ligated into XhoI and XbaI sites of the vector pMAMneo-

blue (CLONTECH), yielding pRABM which failed to express RABM. Using EcoRV

and XbaI, I transferred the RABM code to pcDNA3 (Invitrogen), yielding

pcDNA3RABM (Fig. 2-2). William A. Dunn, Jr. provided a pcDNA3-based vector,

pHAHAB which expresses human aldolase B (Sakakibara et al., 1989) with an amino

terminal 12CA5 HA epitope, HAHAB (Lenk et al., Submitted 1998).






a. WID5, 5' primer: CTCCCTTGGCTCGAGCTGTC
XhoI


Xbal
b. WID9, 3' primer: TGCTCTAGACTActacaagtcttcttcagaaataagcttttgttcctcGTAGG-
TGTAGGGGCTGTGA



Figure 2-1: Primers for PCR Amplification of Insert Containing cDNA for RABM
Expression. a) 5' primer also called, WID5; b) 3' primer also called WID9. Orientations
are relative to 5' to 3' convention. Italics indicate reverse complementary code for
carboxyl terminal amino acids; lower case letters indicate reverse complementary
sequence for myc (9E 10) tag; single underline indicates reverse complementary sequence
for a stop codon; boldface indicates XhoI and XbaI restriction sites; other bases are the
same as vector sequences.










a. 5' DNA Sequence:


--TCTGCAGATATCAAGCTTA TCGA TA CCGTCGACCTCGAGCTGTCAATCAIT.--
EcoR V XhoI start
methionine
(aldolase B)



b. 3' DNA Sequence:

myc-epitope code
--ACCTACgaggaacaaaagcttatttctgaagaagacttgTAGTCTAGAGGGCCC--
C-terminal stop XbaI
tyrosine codon
(aldolase B)



C. Carboxyl terminal amino acids of RABM:

--TASYTYEEOKLISEEDL



FIGURE 2-2: DNA Sequence ofpcDNA3RABM. a) 5' insertion site and b) 3' insertion
site showing new sequence generated by vector construction. Boldface designates
cDNA sequence of rat aldolase B (upper case) and human myc epitope 9E10 (lower
case). Italics designate DNA sequence from the multicloning site ofpMAMneo-blueTM.
Standard typeface designates DNA sequence in the multicloning site ofpcDNA3TM
(Invitrogen). Underline designates indicated restriction sites. Double underline
designates indicated codons. Sequences not shown for rat aldolase B cDNA and
pcDNA3TM vector are available in Tsutsumi, et al. (Tsutsumi et al., 1984) and from
Invitrogen Technical Services, respectively, c) Amino acid sequence predicted for the
carboxyl terminus of the RABM protein expressed from this vector (residues not shown
would be identical to rat aldolase B), single letter amino acid code is underlined for
residues added to create a 9E10 epitope.










Site-Directed Mutagenesis of pcDNA3RABM

I used three different strategies for PCR mutagenesis: (1) overlap extension (Ho

et al., 1989), (2) Quick-ChangeTM Site-Directed Mutagenesis Kit (Stratagene #200518),

or (3) restriction-limited insertion as described below (Fig. 2-4; Table 2-1).

I used an overlap extension protocol adapted by Brian Cain from Ho and others

(Ho et al., 1989). In brief, a mutagenic primer pair (see Fig. 2-3, positions 2 & 3, 4 & 5,

or 6 & 7) was made complementary to each other and to base pairs on either side of a

targeted change (non-complementary) in aldolase B coding sequence (Table 2-1). For

example, Q58 mutagenesis started with pcDNA3RABM as template in two PCR

reactions using primers at positions 1 & 2 (Fig. 2-3) to make product for the 5' end of an

insert and at positions 3 & 8 (Fig. 2-3) to make product for the 3' end of an insert. PCR

was done for 35 cycles (1 min. at 94C, 1 min. at 55C, and 2 min. at 72C).

At one end of one product, sequence was derived from primer at position 2 and

therefore was reverse complementary to one end of the other product derived from

primer at position 3. These products were combined in Taq polymerase buffer

(Promega), melted at 94C, and cooled very slowly to allow promiscuous annealing and

yielding a small amount of 3'end-to-end annealed single stranded DNA from 5' and 3'

products for ends of a desired insert. Taq polymerase was added and the temperature

raised to 72C for run-off extension through ends complementary to positions 1 & 8

(Fig. 2-3). Primers for positions 1 & 8 were added and cycled through temperatures as

done previously which produced a smear of products due to promiscuous annealing.













Rat AldolaseB


1 3
4^I 1'3


5 7


I I -- -- -- --
B E 2 4 6 B b


FIGURE 2-3: Positions of Primers for Site-Directed Mutagenesis. Map shows the RABM coding region
ofpcDNA3RABM; positions drawn to scale. Numbered arrows identify 5'-> 3' primer sequences at
complementary sites. Shaded areas are cDNA sequences for the indicated polypeptides. Letters B, E,
and Xb indicate restriction sites for enzymes BsaI, EcoR V, and Xba I, respectively. E and Xb are sites
of insertion into pcDNA3 (Invitrogen) multicloning site.


TABLE 2-1: Details of Primers for Site-Directed Mutanenesis


Position (bp)
Primer Map From Target Primer Sequence, 5'to 3'
I.D. Posi- 1St Base of Code (sequence non-complementary
_____ tion* Start Codon Change to pcDNA3RABM is underlined)
WID17 1 -117 to +SmaI CTCACTATAGGGAGACCCGGGCTTGGT
-91______________
WID19 2 +25 to Q12(T/N) CTCCTTrCTTA(T/G)TCTCTGAGGT
+45______________
WID18 3 +25 to Q12(T/N) ACCTCAGAGA(A/C)TAAGAAGGAG
+45
WID21 4 +163 to Q58(T/N) CTCGGAA'/G)TCCTTCGGT-
__________+181___________
WID20 5 +163 to Q58(T/N) AACCGAAGGA(A/C)ITCCGAG
__________+181____________
WID44 4- +165 to +ScaI AGAGAAGTACTAAAGAGGAGCTCTCGG
non*** +195 Q58N AAATTCCTTCGG
WID43 5- +196 to +PmlI CGAGACACGTGGACAATTCTATC
_____non*** +218
WID23 6 +322 to Q11 1(T/N) ACCTCCA(GT)TGTCCAGCTT
~________+339_____________
WID22 7 +322 to Q111 (T/N) AAGCTGGACA(A/C)TGGAGGT
______+339 ______________
WID36 6 +313 to QUIT CACCTCCTGTGTCAAGCTTGATGCCCAC
+340 +HindlII___________
WID35 7 +313 to +HindIII GTGGGCATCAAGCTTGACACAGGAGGTG
_________+340 Q1I1TI
WID24 8 +617 to none AGCAGCCAAGACCTTCTCAG
________+636 _______________________________
*Fig. 2-3;**amino acid (single letter code) change by residue # (start M = 1); -**primer non-
complementary to paired primer with position shifted for restriction-limited insertion (Fig. 2-4).








40
Though extraneous products were common, the most abundant product was the desired

mutated DNA fragment representing full length mutated insert. This insert containing

altered DNA code was BsaI digested, gel purified, and ligated into the corresponding

site of fresh pcDNA3RABM from which the wild type fragment was removed.

Normally, restriction sites for primer positions 1 & 8 would be designed for two unique-

site enzymes producing different overhangs. However, BsaI cuts outside its recognition

sequence producing randomly unique overhangs that abrogate a need for separate

enzymes. BsaI cuts a third site near the ampicillin-resistance gene of pcDNA3RABM,

producing two fragments of vector besides the insert fragment. Overhangs for all three

sites were randomly different allowing three-fragment ligation with proper orientations.

The Quick-ChangeTM Site-Directed Mutagenesis Kit (Stratagene) was also used

and found to be more rapid. The manufacturer's protocols were followed, and primers

designed for primer extension and amplification at QI 11 (WID23 and WID22 at

positions 6 & 7) did not work with the Quick-ChangeTM kit. However, longer primers

(WID36 and WID35) were successful. The Quick-Change protocol involves in vitro

synthesis of the entire vector (6.5 kb), possibly introducing errors anywhere in mutated

pcDNARABM. To reduce sequencing, the BsaI fragment containing new mutations was

cassette into fresh vectors. All altered regions of vectors were sequenced at least twice

to confirm changes in amino acid coding were specific for targeted residues.

For Q58N mutation, a restriction-limited insertion was designed(Fig.2-4). This

method uses a mutagenic primer (WID44) to span the Q58 codon and code for unique










blunt-end restriction site, Scal (restriction-limited). Another primer (WID43) was

designed with another unique blunt site, PmlI, such that ScaI to Pml I blunt ends ligated

to make proper aldolase B code. Steps were followed as indicated in Figure 2-4 to

produce an expression vector for the mutated RABM, pcDNA3RABMQ58N.

Expressing Epitope-tagged Aldolase B in Cell Lines

Permanent Lines Expressing RABM

E36 and ts20 cells were transiently transfected with pcDNA3RABM, using Lipofectin

(GIBCO BRL) or DOTAP (Boehringer) by the manufacturers' protocol. When

transfected cultures approached confluency labeled cells occurred in groups of 2 to 8

presumably due to cell division. Transfected cultures were trypsinized at confluency and

diluted >1:15 by area into G418 Medium. Fresh G418 Medium was provided every 1-2

days as needed to remove cell debris and maintain strong selection. By 2-3 weeks post

plating, colonies of resistant cells were isolated, passage, and screened for RABM

expression immunofluorescencee microscopy and western blotting with anti-9E10

monoclonal antibody as described below).

Different lines permanently expressed RABM at varied levels (-10 fold range on

western blots). Using experimental protocols described below, no effect was seen with

doubling time, degradation of RABM, degradation of total protein, or viability. Of eight

positive clones that continue to express after culture amplification, seven (4 from E36

and 3 from ts20) maintained stable relative levels of RABM for twenty additional

continuous doublings or longer (by immunofluorescence and on western blots, data not

















target codon
for Q58
I


WID5 | UjUAUAAlN.Ai. -0
Xho Il I
X1 GGCTTCC TAAAGGCTCTCGAGGAGAAATCAT C
W ED44 / AGAGA
I Sca 5'


1. PCR with WID5 and WID44
2. Digest with Xho I and Sca I


TCGAGCTT A O'_ITTTAGT
CGA^^' AAAICA GTGGACA'
'AGCT CACCTGT

3. Ligate to make pcDNA3RABMQ58N


1. PCR with WID44 and WID9
2. Digest with Pml I and Xba I


/1 TACACrC"N%


pcDNA3RABM digested with Xho I and Xba I


Figure 2-4: Restriction-Limited Insertion for Constructing pcDNA3RABMQ58N.
Above, primers (small arrows and sequences for WID43 and WID44) are shown relative
to pcDNA3RABM template (bar). Sequences juxtaposed to template indicate
complementary regions. Relative positions of Q58 codon and restriction sites are
indicated. Below, DNA pieces for a three-fragment ligation. See text, Fig. 2-3, and
Table 2-1.


Pm] I
5 GAA/ W3D43
AG .~c/ .. ..... '


' pcDNA3RABM
or template
wMx









43
shown). To facilitate experimental quantification, the highest RABM-expressing E36

and ts20 cell lines fully designated E36RABM14.1 and ts20RABM10.2 or abbreviated

throughout this dissertation as E36AB and ts20AB, respectively, were used. In control

experiments, variation in the level of RABM expression did not affect results (data not

shown).

Similar procedures were repeated with Fao rat hepatoma cells transfected, except

another plasmid pHAHAB also was used to express human aldolase B tagged with the

12CA5 HA epitope at its amino terminus (HAHAB). Screening for HAHAB expression

was done with monoclonal antibody against 12CA5. When G418-resistant clones were

isolated at very most 20% of cells in a given clone had visible expression that was mostly

dim with a few bright cells, and this fraction was rapidly lost with culture splitting for

amplification. Subcloning and screening of a few hundred colonies produced one truly

stable line expressing HAHAB at levels comparable to ts20AB expression of RABM.

This line was designated FaoAB. FaoAB cells split at low density (1:20 or less) grew

much slower than parent Fao cells. In passages using about 1:10 or 1:15 splits, growth

rates were similar to parent Fao cells. An attempt to isolate HuH7 cell lines expressing

RABM was made, but failed to produce any clones having permanent expression.

Transient Transfection System

Fao, HuH7, NRK, and BHK (hepatic and renal cell lines derived from tissues that

express aldolase B) were transfected for transient RABM expression with a series of

lipids according to manufacturer's standard protocols (Invitrogen and Boehringer-









44
Mannheim). Transient transfection gave very broad cell-to-cell variation in expression by

9E10 immunofluorescence, but ratios of bright to dim cells were relatively reproducible

between transfections. Transfection efficiency was defined as fraction of labeled cells.

Immunofluorescence

Cells were grown on glass coverslips to desired confluency, rinsed briefly with

PBS and fixed with 4% paraformaldehyde in PBS for 20-30 minutes. Fixed cells were

washed three times for 10 minutes in 50 mM ammonium chloride/ 0.1% Tx-1 00/PBS.

Coverslips were placed on drops containing antibody diluted 1:100 in 5%NGS/ 0.1%

Tx-100/PBS for 1-2 hours at room temperature. Coverslips were washed four times for

5 minutes in 0.1% Tx-100/PBS. Coverslips were placed on drops containing an

appropriate secondary antibody (rhodamine or fluorescein conjugated) diluted 1:100 in

5%NGS/ 0.1% Tx-100/PBS for 1 hour at room temperature. Coverslips were washed

six times for 5 minutes in 0.1% Tx-100/PBS, then mounted on Fluoromount G (GIBCO

BRL).

Antibodies

Preparation of Ubiquitin-Free Aldolase B Antigen

Different E. coli strains were transformed with pXPB, a plasmid vector kindly

provided by Dean R. Tolan for bacterial expression of enzymatically active human

aldolase B (Beemrnink and Tolan, 1992). The aldolase B expressed in E. coli retains all

the enzymatic properties of the protein isolated from human liver (Sakakibara et al.,









45
1989). Increased expression of a 40 kD Coomassie signal in SDS-PAGE of whole cell

preparations was apparent in transformed cells.

E. coli JM83 cells were transformed with pXPB, which produced much more 40

kD protein than untransformed cells, about 0.3 mg per ml of 1.9 OD600nm culture. A 250

ml culture of LB broth + ampicillin (10.tg/ml) was grown to 1.9 OD6o0 nm culture and

pelleted in a Beckman GSA rotor at 3500 rpm for 10 min. Samples were maintained at

0-4C for the rest of the procedure. The cell pellet was resuspended in 20 ml 15%

sucrose/50 mM EDTA/50 mM Tris-HCl pH 8.5. To this, 5 ml 5 mg/ml lysozyme was

added, gently mixed by inverting, and incubated 15 min. Then 15 ml 0.1% Triton X-

100/50 mM Tris-HCl pH 8.5 was added, gently mixed, and incubated with periodic

inverting for 20 min. After centrifugation at 9000 rpm for 30 min. (GSA rotor), the

supernatant was decanted into Polyclear tubes and ultracentrifuged in aSW27 rotor at

23,000 rpm for 60 min. The resulting supernatant was the crude extract which was

further processed as previously described for isolation of aldolase B from liver extracts

(Penhoet and Rutter, 1975). Saturated (NH4)2SO4 was slowly added (0.5 ml/min.) to

45% final concentration with constant stirring. After centrifugation at 9000 rpm for 60

min., the supernatant was collected and the pellet discarded. (NH4)2SO4 was slowly

added to 60% concentration, and 6 N NH4OH added to pH 7.5. The mixture was

immediately transferred to centrifuge bottles and let stand for >2 hours. After

centrifugation at 9000 rpm for 60 min., the 60% (NH4)2SO4 pellet was dissolved in 1

mM EDTA/10 mM Tris-HCl pH 7.5 and dialyzed against the same buffer. The sample










was loaded onto a 25 ml phosphocellulose (fine mesh, 1.26 meq/g) column prepared

exactly according to Penhoet and Rutter. The column was washed with 5 mM EDTA/50

mM Tris-HCl pH 7.5 (50-60 ml) until OD280 m approached zero. Then 2.5 mM fructose

1,6- diphosphate in 1 mM EDTA/10 mM Tris-HCl pH 7.5 was used to specifically eluted

a sharp aldolase peak. Peak fractions with specific activities of 0.83 to 0.98 aldolase

U/mg (1 U/mg expected for aldolase B) were pooled, precipitated by 55% (NH4)2S04,

and stored as a suspension at 4C. Before immunization, the suspension was dialyzed

into 10 mM Tris-HCl pH 7.5. According to Coomassie labeled SDS-PAGE and

enzymatic properties (Rutter et al., 1966), the recombinant human aldolase B constituted

at least 95% of the final protein and was more than 99.99% pure of bacterial aldolase

activities, containing <0.005% bacterial isoform (EDTA-sensitive) activity.

Production of Antibodies Against Aldolase B

Rabbits were fed and housed by University of Florida Laboratory Animal

Services. Antibodies to native and denatured aldolase B were raised as previously

described (Reznick et al., 1985) except ubiquitin-free human aldolase B antigen was used

(as prepared above). For making antibody to native aldolase B, 50 gg antigen in 0.5 ml

10 mM Tris-HCl pH 7.5 was emulsified 1:2 with complete and incomplete Freund's

adjuvants for immunizations and boosts, respectively. For antibody to denature antigen,

the antigen solution was supplemented with 2%SDS/2%O3ME and boiled 10 min. prior to

mixing with adjuvant and immediately before administration to animals. Intradermal










injections were in the thoracic region on the backs of specific pathogen-free New

Zealand White rabbits. The first boost was two weeks after initial immunization.

Preparative Western blots of rat liver cytosol were routinely used to follow

specific immunoreactivities. One week after the first boost, the rabbit receiving native

antigen produced a highly reactive serum specific for 40 kD aldolase B which was

maintained without further boosts. Unless otherwise specified, these antibodies were

used to detect native aldolase B-specific polyclonal epitopes throughout this study.

One week after the first boost, the rabbit receiving denatured antigen produced

antibody that specifically labeled a 68 kD protein moderately, 60 kD and 78 kD proteins

lightly, and a high molecular weight smear. This pattern was remarkably similar to that

for anti-Ub68. Furthermore, relative recognition ofisoforms A and B were comparable

to that for Ub68. Thus, denaturation of aldolase B disrupted isoform-specific epitopes

and produced anti-denatured aldolase B that similarly recognized epitopes in both

aldolases A and B. Interestingly, the early bleeds had little or no reactivity to 40 kD

aldolase.

Every 4-7 weeks (after injection sites completely healed), the rabbit immunized

with denatured aldolase B was boosted. After the second boost, antibody

immunoreactivities were greatly increased. Though these sera recognized some non-

protein epitopes, they retained specificity for proteins labeled with sera from earlier

bleeds. After the third boost, sera recognized 40 kD aldolase B but never as well as

antibody to native antigen.










Other Antibodies

Anti-Ub68 was provided by William A. Dunn, Jr. and is a polyclonal rabbit

antibody raised against a major ubiquitin-protein conjugate purified from lysosomes

(Lenk et al., Submitted 1998). Monoclonal mouse antibodies against thel2CA5 HA and

9E 10 myc epitopes were obtained from the University of Florida Hybridoma Core

Laboratory. Alternatively, hybridoma cells expressing anti-9E10 (provided by the

Hybridoma Core) were injected into the peritoneum of BALB/c mice (provided by the

University of Florida Laboratory Animal Services Division), and periodically, ascites was

harvested until mice showed signs of discomfort or disease at which time they were

euthanized.

Viability Assays

All treatments were measured for viability except for incubations in HBSS

(Hank's Balanced Salts Solution) which were used to induce cell death. Heat stressed

cultures using HBSS had many cells rounding and sloughing off culture surfaces in 4 to 6

hours of treatment, and were only used in experiments to determine the effect of dying

cells on protein degradation measurements. When HBSS was replaced with MEM

(Sigma #M-0268 + 2.2 g/1 sodium bicarbonate), such rounding and sloughing was

delayed beyond 25 hours of treatment. To measure metabolic viability, cultures exposed

to each experimental treatment were recovered under normal maintenance conditions for

12 hours followed by addition of the same labeling medium (0.1 mCi 35S-methionine/ml)

used for protein degradation assays. Then protein synthesis was measured as TCA










precipitable counts incorporated in 20 min. Metabolic viability was defined as the %

protein synthesis relative to duplicate cultures treated with fresh medium under normal

maintenance conditions. Since experimental treatments (see Experimental Conditions)

lack serum and nutrients (MEM instead of DMEM) relative to maintenance conditions,

our assay probably underestimates metabolic viability. In MEM, more than 80% of

metabolic viability was retained for 28 hours and 15 hours in heat stressed E36 and ts20

cells, respectively. Unless otherwise stated, all data are reported for incubations and

drug treatments that retained 90% or greater metabolic viability. For low density

cultures (<10% confluent), the Cell Titer 96 AQ System was used according to

manufacturer's protocols (Promega) which indirectly measures electron transport

pathway activity.

Subcellular Fractionation

Subcellular fractionation of E36 and ts20 cells is summarized in Figure 2-6. To

produce a homogenate containing intact organelles, cells were grown to recent

confluency in 100 mm dishes, using 10 ml DMEM (Sigma #D-5648) + 10% FBS

medium. Culture rinsed with 10 ml DPBS (Sigma D-5652) was treated with 1.0 ml 1 X

Trypsin-EDTA (Sigma #T-9395 diluted in DPBS),and incubated at room temperature

until cells easily and completely knocked loose from the plate (10 minute maximum).

After adding, 5 to 10 ml medium containing 10% FBS, cells were transferred to 15 ml

conical centrifuge tubes (polypropylene), centrifuged at 1,500 X g for 5 minutes, and

supernatant discarded. Pellet was suspended in 1.0 ml ice-cold cavitation buffer (SHE):







Figure 2-6: Subcellular Fractionation Scheme for E36 and ts20 Cells. 50


Cell Culture


Scrape cell sheet

Centrifuge 1000 x g


-> Supernatant = Sc


Pellet


Homogenize
4


--> Homogenate = Ho


Centrifuge 1000 x g
$
Supernatant

Centrifuge 1000 x g
S
Supernatant = L
4-


Centrifuge 100,000 x g
4,.
Pellet
4,.
Resuspend

Centrifuge 100,000 x g


-> Pellet
4-
Pool = LP
T
-* Pellet


-> Supernatant = HS


-> Pellet = HP


Supernatant (wash, dilute HS) --> <1% of culture content








51
250 mM sucrose, 10 mM HEPES (Research Organics #6003H-3), 1 mM EDTA, pH 7.4,

loaded into a N2-cavitation bomb chamber using 2-3 ml total SHE volume, pressurized

to 65 psi for 10 minutes, and collected sample from bomb directly into a pre-chilled

Dounce homogenizer. Procedural details were as described by the cavitation bomb

manufacturer's specifications (Kontes). The sample was homogenized with 5 strokes of

a pestle, minimizing froth by limiting passage of bubbles to the sample side of pestle.

This was saved as the homogenate (Ho).

Alternatively, the trypsinization step was replaced by scraping the cell sheet

directly into SHE which resulted in a large fraction of cytosol but not organelles to leak

out. This facilitated the separation of aldolase associated with organelles from soluble

aldolase in the cytosol. Scraped cells were pelleted as done above to remove trypsin

solution, but in this case the supernatant was saved (to assess cytosolic leakage) as the

scrape fraction (Sc). The rest of cavitation and homogenization was performed as

above.

Homogenate (Ho) was fractionated essentially as described previously

(Rickwood, 1992; Coligan et al., 1995), using Sigma reagents for assays and Sorvall or

Beckmann centrifuges and accessories. Homogenate was centrifuged at 1,500 X g for

20 minutes yielding a low speed pellet (LP) and supernatant. The centrifugation was

repeated with the supernatant to make sure nuclei, large debris, and unbroken cells were

efficiently removed, the resulting pellet was pooled in LP, and the resulting low speed

supernatant (LS) was centrifuged at 100,000 X g for 90 minutes, yielding a high speed









supernatant (HS) and pellet. The pellet was resuspended in well over 400 volumes of

fresh SHE and centrifuged at 100,000 X g for 60 minutes. The supernatant had a

content similar to HS but was much more dilute (data not shown), so it was not pooled

with HS. The pellet was saved as the high speed pellet (HP).

Fractionation conditions were developed to separate abundant cytosolic aldolase

from that associated with organelles. An initial scrape into fractionation buffer caused a

three fold greater leakage of aldolase than acid phosphatase, so this step was retained in

the procedure. Conditions were chosen to maximize recovery oflysosomal organelles

(acid phosphatase and 3-hexosaminidase) from LP to HP and minimize release of

lysosomal markers into Sc and HS. Recovery was reasonable (85-99% accounted).

Enzyme Assays

Aldolase

Aldolase assays were performed as previously described (Penhoet and Rutter,

1975). "Aldolase reaction mix" includes 50 p1 6.3 mg/ml a-glycerophosphate

dehydrogenase-triose phosphate isomerase mixture + 4 mg NADH (99% pure, Sigma) +

20 ml 0.1 M glycylglycine pH 7.5. Add 5-50 pl of sample to 1 ml of aldolase reaction

mix, and measure background AOD (340 nm)/min. (BG); add 50 pl 50 mM fructose-1,6-

diphosphate (FDP) or 100pl 100 mM fructose-I-phosphate (F-l-P), and measure assay

AOD (340 nm)/min. (ASSAY). Aldolase activity in units, U = (ASSAY-BG)/12.44 for

FDP or = (ASSAY-BG)/6.22 for F-i-P.










Acid Phosphatase

Acid Phosphatase activity was the OD (405 runm) in 1000x g supernatant after 60

minute incubation at 37C for 50 ul sample in 200 pl 8 mM p-nitrophenolphosphate/

2mM MgC12/ 90 mM Na-acetate pH 5.0 stopped by 600 ul 0.25 M NaOH (Rickwood,

1992).

Protein Analysis

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), gel

staining and drying, western blotting, and autoradiography followed standard protocols

similar to those described and referenced elsewhere (Ausubel et al., 1994; Coligan et al.,

1995). Gels were made with 10% polyacrylamide (1:35 bis-acrylamide).

Immunochemiluminescent detection was done with Amersham ECL Reagents and

protocols, except blots were rinsed in Kodak 1XCDS buffer just before exposure to ECL

chemicals. Protein concentrations were determined with Bio-Rad protein assay (IgG

standard) reagent or spectrophotometric absorbence (205 nm or 280 nm).

For direct immunoprecipitations, monoclonal antibody was purified and

conjugated to sepharose 4b by a CNBr protocol similar to that described in Coligan et

al., 1995. Cells were incubated on a shaker at 0-4C with ice cold minimum lysis buffer

(MLB: 1%NP40/ 1 mM EDTA/50mM Tris-HCl pH 7.4), standard lysis buffer (SLB:

150 mM NaCl MLB), or modified radioimmunoprecipitation assay buffer (mRIPA:

0.1% SDS/ 0.25% desoxycholate/ SLB) supplemented with a cocktail ofprotease

inhibitors (leupeptin, TLCK, pepstatin, aprotinin, and PMSF obtained from Sigma or










Boehringer/Mannheim) at concentrations according to Harlowe. The mRIPA gave the

best results with mAb's conjugated to sepharose, and SLB gave the best results with

polyclonal sera precipitated with commercial protein A-agarose (Boehringer-Mannheim).

Lysates were precleared for 15-20 minutes on maximum in a microfulge then transferred

to tubes containing 4-10 ll packed bed volume of mAb-sepharose or 1-5 p.l antiserum

then rotated in the cold. After one hour, tubes containing antiserum only received 10 gl

packed bed of protein A-agarose. Rotating incubation was continued for varying times,

usually overnight, and such times were held constant within a given experiment. Rapid

washes by quick microcentrifugation and aspiration of supernatant were done three

times with the same lysis buffer and one time with TBS, then immediately processed for

other procedures. For SDS-PAGE, a 2-fold concentration of sample buffer was directly

applied to the pellet and heated at 95-100C for 5-10 minutes. For precipitable aldolase

activity, pellets were further washed with TE pH 7.5 then resuspended in aldolase

reaction buffer.

Densitometry autoradiographss, chemiluminographs, & Coomassie gels) were

quantified using a desktop scanner and Sigmagel software. Automatic brightness and

contrast settings determined initial settings that were then kept constant. Spot settings

were chosen to encompass regions of interest, reduce background, and optimize signal.

Standard curves were routinely performed to characterize linear response ranges for

relative protein levels which also defined backgrounds and allowed quantification of

relative signals. Where protein bands were specific for transfected cell lines,










untransfected cells were run in parallel through procedures and quantified to establish

backgrounds.

Stress-Induction of Protein Degradation

Culture Preparation

Cells were plated, grown, and maintained at confluency for 2-3 days. About 30-

40 hours before an experiment, cultures were fed with standard maintenance medium or

for protein degradation assays, 14C-Valine or "3S-Methionine was made up in comparable

medium with the corresponding unlabeled amino acid omitted. At the beginning of an

experiment, cells were switched to the media and temperatures indicated in Table 2-2.


Table 2-2: Comparison of Systems for Stress-Induced Degradation of Proteins
Heat Stress Induction Starvation Induction
MduControl Stress Control Stress
Medium MEM MEM DMEM+FBS KH*
Temperature 30.5C 39.5C 37C 37oC
*KH, Krebs-Heinseleit medium (Lefer et al., 1982)

Heat Stress

Cultures were prepared as described above and replicates were incubated under

control temperature (CT) and heat stressed (HS) conditions (Table 2-3) which are

permissive and non-permissive, respectively, for the ubiquitin-activating enzyme El of

ts20 cells (Handley-Gearhart et al., 1994; Kulka et al., 1988; Lenk et al., 1992).

Accordingly, CT conditions included 4 mM bicarbonate-buffered MEM under 5% CO2 at

30.5C, and HS conditions included 20 mM HEPES-buffered MEM under air at 41.5C

for 1 hour followed by 39.5C. These incubations followed established protocols for the









E36/ts20 cell system, except MEM replaced HBSS to improve viability. In some

experiments, media were supplemented with 5-10 mM 3-methyladenine (3MA) or 40-

160 .M chloroquine (CHQ) as indicated in Results. Logistics and consistency with

published protocols required differences in buffering and atmosphere between CT and

HS conditions. As a control for such


Table 2-3: Heat Stress (HS) and Control Temperature (CT) Treatments
to Determine Ubiquitin-Activating El Mediated Processes
Experimental
Condition MEM buffer* Temperature Incubator

CT 2.2 g/ 1 NaHCO3 30.5C standard
(Permissive) pH 7.4 5% CO2


HS 20 mM HEPES 1 hour @ 41.5C submerged
(Non-permissive) pH 7.4 then 39.5C in water bath

Repeat treatments with two cell lines: E36 (parent) and ts20 (mutant)
*For protein degradation experiments, medium was supplemented with unlabeled amino
acid corresponding to that used for labeling (5 mM methionine or 10 mM valine).
differences, CT as summarized in Table 2-3 was compared with CT in HEPES-buffered

MEM under air, yielding no differences in cell morphology, viability, or protein

degradation measurements (data not shown). The other control (i.e. comparing HS in

conditions above with HS in bicarbonate-buffered MEM under 5% C02) was not tested,

because bicarbonate buffering varies with temperature.

Starvation (Nutrient Stress)

Cultures were prepared as described above and replicates were refed with fresh

standard maintenance medium (DMEM + 10 % FBS) or Krebs-Heinseleit (KH) medium









57
and referred to as "Fed" or "Starved," respectively. KH components are given in Lefer,

et al., 1982. For protein degradation experiments, media were supplemented with

unlabeled amino acid corresponding to that used for labeling (5 mM methionine or 10

mM valine). Inhibitor treatments utilized the same levels as for heat stress above.

Protein Degradation

Permanent cell lines or transiently transfected cultures were treated according to

instructions in the section Stress-Induction of Protein Degradation to produce cells with

metabolically labeled proteins containing "S-methionine or 14C-valine. Cell sheets were

routinely rinsed with DPBS (Sigma) immediately followed by application of media

containing unlabeled excess amino acid (5 mM methionine or 10 mM valine) to cultures

described above. This initiated chase of radiolabel incorporated into proteins.

At various times, aliquots of media were collected and TCA precipitated to

measure release of soluble counts measured with a scintillation counter. At the end of

the chase, whole cultures were TCA precipitated to determine total counts. Fraction of

TCA soluble counts released at various times were subtracted from 1 to calculate TCA

precipitable counts, representing the remaining total radiolabeled protein at those times.

Alternatively, cultures were harvested at each time point, processed for

immunoprecipitation, SDS-PAGE, and autoradiography, and the radioactive signals in

specific protein bands (absent in untransfected cells) were quantified by densitometry,

representing the remaining radiolabeled protein (aldolase) at those times.








58
To compare degradative rates for proteins expressed at different concentrations,

relative rates are normalized to the initial amount of the protein, giving a fractional

change per time with units %/hour. This fractional rate is constant for unchanging

degradative mechanisms regardless of a substrate protein's concentration. For a given

protein, this fractional rate defines the degradative rate constant, kd. For total proteins,

the fractional rate represents weighted average kd contributed by the varied amounts of

different proteins. Throughout this dissertation, all degradative rates and other rates of

protein decrease are estimated with the following calculation. Fractions of radiolabeled

protein remaining were transformed by the natural log (In) and regression analysis

performed using the following function:


ln(100*St/So) = -kd t,


where St = signal at time t, So = initial signal, kd = first order degradative rate constant,

and t = time. Degradation rates were taken as the negative slope of the regression (kd)

and the standard error of the slope was calculated as the standard error ofy at x divided

by the square root of the deviations squared ofx. Degradative turnover is also described

by half-life, t%, the time needed to replace 50% of existing molecules with new ones. In

general, the degradative rate constant and half-life are simply converted by kd = ln(2)/ ty

= 0.693/ t. Note that some investigators do not follow the empirically confirmed first

order relationship of kd and ty. This results in kd and t% reported in the literature that can

vary by as much as three fold from similar data presented using conventional

calculations.















CHAPTER 3:
UBIQUITINATION MEDIATES LYSOSOMAL PROTEOLYSIS OF ALDOLASE B


Introduction

Aldolase B undergoes degradation during stress via autophagy. In Chapter 1,

evidence was described for ubiquitin protein conjugates which underwent starvation-

induced enrichment in autophagic vacuoles and lysosomes, and preliminary work

suggested that putative ubiquitinated aldolase B was amongst these conjugates (Figs. 1-3

and 1-4). This resulted in the hypothesis that stress-induced autophagic degradation of

aldolase B requires ubiquitination. In the first part of this chapter, the presence of

aldolase B in ubiquitin conjugates is confirmed, and a role for ubiquitination in the heat

stress-induced autophagic degradation of aldolase B is demonstrated.

Two mechanisms were found to simultaneously mediate enhanced degradation of

aldolase B during heat stress: autophagy and cytosolic proteolysis. To separately

examine effects on autophagy, heat stress-induced changes in endogenous aldolase A and

exogenous epitope-tagged aldolase B associated with pelletable organelles were assayed.

In addition, autophagic degradation of long-lived proteins was demonstrated to require

ubiquitination. The results described below support a role for ubiquitination in the

function of a subset of lysosomal proteases.










In Vivo Multiubiquitination of Aldolase B

Previously, antibodies were raised to a major ubiquitin-conjugated protein, Ub68,

that associated with autophagic vacuoles and lysosomes during stress-induced

autophagy. On western blots of subcellular fractions isolated from rat liver, anti-Ub68

produced a pattern suggestive of a 40 kD protein successively conjugated with serially

increasing numbers ofubiquitin. Such a pattern is referred to as a ubiquitin ladder for

the modified core protein. Peptide sequence analysis identified the 40 kD protein as

aldolase B, suggesting that anti-Ub68 reactive proteins might represent a ubiquitin ladder

for aldolase B.

To confirm the existence of ubiquitinated forms of aldolase B, antibodies (Fig. 3-

2) were raised against aldolase B (Fig. 3-1) and used to assay western blots of rat liver

fractions from the previous studies (Fig. 3-3). In short, anti-aldolase B recognized the

same proteins as anti-Ub68, confirming a ubiquitin ladder for aldolase B and

demonstrating that aldolase B is multiubiquitinated in vivo. The data are consistent with

ubiquitin-mediated autophagic degradation of aldolase B.

First, antibodies against aldolase B had to be produced. However, aldolase B

isolated from animal cells would need to be purified away from contaminating ubiquitin

and ubiquitin conjugates. Since bacteria lack ubiquitin, aldolase B was expressed in E.

coli and purified to produce ubiquitin-free antigen for immunization (Fig. 3-1). E. coli

strains transformed with an expression vector for human aldolase B (Fig. 3-la, lanes x)

produced more 40 kD protein (expected size of aldolase B) relative to untransformed

cells (Fig. 3-la,



















Figure 3-1: Isolation ofUbiquitin-Free Aldolase B Expressed in E. coli. a) Three E. coli
strains DH5c, JM109, and JM83 were transformed for aldolase B expression as
described in the text, pelleted, suspended in sample buffer, boiled, and run on SDS-
PAGE with 60 la culture (OD600 n = 1.5) equivalents per lane, u and x designate
untransformed and transformed cells, respectively. The 40 kD band specific for
transformed cells is indicated; b) E. coli JM83 cells expressing aldolase B were
fractionated as described in the text: 1 and 2, whole cell preparations (as in part a)
untransfected and transfected, respectively; 3, crude extract; 4, 45% (NH4)2S04
supernatant; 5, 45-60% (NH4)2SO4 cut; 6, aldolase activity peak from phosphocellulose
column; 7, dialyzed antigen ready for immunization; 8, rat liver cytosol used to screen
antibodies; 9, detection of lane 8 with antibody raised against protein in lane 7. Lanes 3
to 6 were loaded with 12 jig, lane 7 with 4 ug, lane 8 with 25 ug, and lane 9 with 10 uLg
of protein. Dark bands on light background indicate Coomassie R-250 label in gels, and
light bands on dark indicate western blotted proteins detected with anti-native aldolase B
by ECL (Amersham); c) Elution profile for phosophocellulose chromatography, Relative
Amounts: Protein, OD(280 nm); Aldolase Activity, mU/10 (loaded then started washes
when collecting fraction 9); FDP 4,, elution started with fructose 1,6-diphosphate.









DH5a JM109 JM83
a. u x u x u x


b. 1


2 3 4 5 6 789


H :. :-


40 00- I
O ---s f


5
4.5
4
3.5
3
2.5 -
2
1.5 -
1-
0.5
0


F jP )
"-L : I


0 5 10 15 20 25 30 35 40 45


Fraction Number


t -- Protein v A
--Aldolase Activity |


I


I























Figure 3-2: Antibodies Against Aldolase B. a) A preparative western blot of rat liver
cytosol run on SDS-PAGE (see Coomassie in Fig. 3-6b, lane 8) was prepared and strips
containing approximately 3 tg total protein were probed with sera from rabbits
immunized with native and denatured aldolase B as indicated, each lane contains a strip,
numbers above each lane correspond to bleed numbers, and numbers with arrowheads
indicate molecular weight, b) A Bio-Rad slot-blotting apparatus was used to load 10
ng/slot of antigen indicated by vertical labels (Aldolase A from Sigma), each row was
probed with serum raised against antigen indicated by horizontal labels with increasing
serum dilutions shown at the bottom of the figure, corresponding preimmune sera were
used in rows immediately above and below anti-aldolase B and anti-Ub68, respectively.








Native
0 1 13
a.

68 l

400 **


Denatured
0 1 2 3 4 5 6 7 8 9 10 11 12 13


111 .fl.j


Serum
Preimmune
Aldolase B
UB68
Preimmune
Preimmune
Aldolase B
Ub68
Preimmune


468

440


-


1
200


1 1 1
800 3200 12800


b.

1|
























Figure 3-3: Aldolase B Ubiquitinated In Vivo is Enriched in Lysosomes. a) Preparative
SDS-PAGE of cytosol from starved rat liver was western blotted onto nitrocellulose
then cut into strips with ~3 tg protein/strip, and stained with early antisera against
aldolase B (Fig.3-2a, bleed 4) or Ub68 (bleed 5); b) Aldolase B antisera from bleeds
after booster injections (Fig.3-6a, bleed 10) were reacted with Cy (cytosol strips as
prepared above) or ML (similar strips using a lysosome-enriched fraction instead). N,
antiserum to active native enzyme; No, same as N withlO-fold longer ECL exposure; D,
antiserum to chemically denatured enzyme; I, antiserum to antigen extracted from
polyacrylamide gel slices; and P, preimmune serum. Arrowheads, molecular weights in
kD. Dots on rightmost edge indicate bands at molecular weights higher than expected
for 40 kD aldolase B subunit. Susan E. Lenk provided subcellular fractions defined as
follows: a 1,000 x g supernatant of rat liver homogenate was centrifuged at 6,000 x g;
the resulting pellet was enriched in lysosomes and contained mitochondria (ML); the
6,000 x g supernatant was centrifuged at 100,000 x g yielding a supernatant fraction
referred to as cytosol (Cy).











Cytosol
Aldolase B Ub68
a. N D P I P


681
o


Aldolase B
b N N No No D D
b. CyMLCyML Cy ML



68"0 Zk

'; 0 0't0* *


. .










lanes u). Aldolase B was purified from the most productive strain, JM83, by cellulose

phosphate chromatography (Fig. 3-1c) of a 45-60% ammonium sulfate cut (Fig. 3-lb,

lane 5) from crude lysate (Fig. 3-lb, lane 3). Cellulose phosphate chromatography

separates aldolase B by substrate affinity at the enzyme's active site, allowing

enzymatically active aldolase B to be specifically eluted with fructose 1,6-diphosphate,

FDP (Fig. 3-1c, peak at fraction #30). Aldolase B in peak fractions was at least 95%

pure based on Coomassie stained SDS-PAGE gels (Figure 3-lb, lane 6) and specific

activities ranging 0.95-0.98 U/mg (pure aldolase B = 1.0 U/mg). EDTA resistance of

purified aldolase B activity indicated that contamination by class II bacterial aldolase was

less than 0.005% (data not shown).

Aldolase B antigen described above was used to raise antibodies against native

and chemically denatured aldolase B as detailed in Materials and Methods (Chapter 2).

Preparative Western blots of rat liver cytosol were routinely used to follow specific

immunoreactivities (Fig. 3-2a). Native antigen produced a highly reactive serum specific

for 40 kD aldolase B. Anti-native aldolase B demonstrated minimal cross-reactivity

with aldolase A (Fig. 3-2b). However, this antibody aldolase B effectively recognized

aldolase B from different animal species(Fig. 3-2, a. 40 kD rat aldolase B, b. purified

human aldolase B).

A rabbit immunized with denatured aldolase B produced antibodies (Fig. 3-2a

"Denatured" bleeds 2 through 5) that specifically labeled a pattern indistinguishable from

that for anti-Ub68 on western blots of subcellular fractions from rat liver (compare Fig.








68
3-3, lanes D with Figs 1-3 and 1-4, anti-Ub68). This demonstrated aldolase B epitopes

in previously identified ubiquitin-protein conjugates and confirmed that aldolase B is

ubiquitinated in vivo. The antibody to native aldolase B was specific for the 40 kD

unmodified monomer. However, with ten-fold greater exposure times, even the anti-

native aldolase B detected a ubiquitin ladder (Fig. 3-3b, No). Preimmune sera failed to

label any proteins. .Antibodies raised against native aldolase B are known to be highly

specific (Haimoto et al., 1989). Given this, labeling with anti-native aldolase B provides

even stronger evidence that aldolase B is multiubiquitinated in vivo, and confirms that

Ub68 is probably a stable conjugate of the form: (aldolase B)i(Ub)4.

Multiubiquitinated Aldolase B is Denatured and Enriched in Lysosomes

Aldolase A and B can spontaneously refold to active enzyme after reversible

denaturation treatments (Horecker, et al., 1972; Beemrnink and Tolan, 1996). Anti-

denatured aldolase B preferentially recognized ubiquitinated (> 40 kD) forms, whereas

anti-native aldolase B preferentially recognized the unmodified (= 40 kD) form (Figs. 3-

2a and 3-3). These results indicated that ubiquitination inhibits spontaneous refolding of

aldolase B into native conformations. Reznick and Gershon also raised antibodies

against native and denatured aldolase B (Reznick et al., 1985). Their anti-denatured

aldolase B failed to immunoprecipitate catalytically active enzyme, but it effectively

precipitated smaller peptides resulting from proteolysis. They also found that native

aldolase B antibody failed to precipitate proteolytic fragments, but efficiently pelleted

aldolase B activity (Reznick et al., 1985). The results reported in this study confirm the









hypothesis that native epitopes of aldolase B require three-dimensional conformations

that mask denatured epitopes.

The 40 kD unmodified aldolase B predominantly occurred in cytosol (Cy)

fractions with <10% in lysosome-enriched (ML) fractions (Fig. 3-3b). Consistent with

previous results (Fig. 1-3), ubiquitin-aldolase B conjugates (bands >40 kD) were

enriched in ML fractions with less occurring in Cy fractions (Fig. 3-3b). Taken together,

the data suggest that ubiquitination can provide a mechanism for maintaining aldolase B

in a denatured conformation. This could contribute to enhanced degradation of aldolase

B by making degradative signals more accessible or by making the protein more

vulnerable to proteases.

Heat Stress-Induced Delivery of Aldolase A to Lysosomes Requires Ubiquitination

Above, ubiquitinated aldolase B was confirmed to contribute to ubiquitin

conjugates that are enriched in autophagic vacuoles and lysosomes during nutrient stress

(Figs. 1-3, 1-4, and 3-3). The results suggested a role for ubiquitination in autophagic

degradation of aldolase B. During heat stress, ubiquitin-dependent autophagic

degradation of long-lived proteins has been demonstrated in E36 Chinese hamster lung

cells (Gropper, et al., 1991; Handley-Gearhart, et al., 1994). Our results suggested that

aldolase B was a possible substrate for this mechanism. However, E36 cells express

endogenous aldolase A but not aldolase B (described later). Both aldolase A and B are

established substrates for autophagy (reviewed in Chapter 1), so aldolase A was

examined as a substrate for ubiquitin-mediated delivery to lysosomes during heat stress.










In addition to autophagy, temperature-dependent cytosolic proteolysis

contributes to increased protein degradation during heat stress (next chapter; Hough and

Rechsteiner, 1984). During autophagy, cytosolic proteins, like aldolase A, are

sequestered into organelles (autophagic vacuoles and lysosomes) that can be pelleted by

differential centrifugation. To measure effects specific for the autophagic pathway,

aldolase A activity associated with pelletable organelles was assayed.

To examine a role for ubiquitination in the stress-induced degradation of aldolase

A in lysosomes, a system developed by Schwartz and Ciechanover was utilized for

measuring ubiquitin-dependent degradation of long-lived proteins (Gropper et al., 1991).

During heat stress, E36 cells undergo enhanced autophagic degradation of long-lived

proteins. However, ts20 cells derived from E36 cells harbor a temperature-sensitive

mutation in ubiquitination. Heat stress is non-permissive for the mutation, so

ubiquitination and enhanced autophagic degradation is inhibited in ts20 cells. The

degradative phenotypes were confirmed and are presented at the end of this chapter.

FDP aldolase activity was used to follow endogenous aldolase A, and acid

phosphatase activity was used to follow organelles in subcellular fractions collected by

differential centrifugation as described in Materials and Methods (Fig. 3-4).

Fractionation was optimized to maximize organelles released from cells, indicated by loss

of acid phosphatase from low-speed centrifugation pellets (LP), and to maximize

organellar integrity, indicated by fraction of acid phosphatase retained in high-speed

centrifugation pellets (HP). Acid phosphatase occurs as both an integral membrane











35%
S30%

S25%
020%
S15%
0 10%
S5%
I-
0%


E36 E36 E36 ts20 ts20
CT HS HS+CHQ CT HS
Cell Line and Treatment


70%


0
S60%
S50%
0 40%
I--
" 30%
0
C
. 20%
S10%
U.%
0%-


b. Acid Phosphatase Activity


Sum T



-LBP T1 TT[JT


E36
CT


E36 E36 ts20
HS HS+CHQ CT
Cell Line and Treatment


ts20
HS


Figure 3-4: Ubiquitin-Dependent Association of Endogenous Aldolase A with
Organelles. Aldolase (a.) and acid phosphatase (b.) reported as % total culture activity
(mean SD, n = 3 cultures) for subcellular fractions collected from E36 and ts20 cells
treated for 8.5 h as indicated; CT, control temperature; HS, heat stress; +CHQ, 80 WM
chloroquine; subcellular fractions were collected (Materials and Methods) and are
labeled only on the first set of three bars (E36, CT): LP, low-speed pellet (1000x g); HP,
high-speed pellet (100,000x g); Sum, total pelleted fractions (LP + HP); different from
CT, Student's t-test: *, p <0.06; **, p <0.03; ***, p <0.009; ****, p <0.0008.










protein and as a soluble matrix protein inside lysosomes. For organelles isolated here,

acid phosphatase activity (about 60% of HP) was released by freeze-thaw (data not

shown), indicating that much of it was soluble in E36 cell lysosomes and served as an

adequate indicator of organellar integrity.

E36 cells were incubated at control (CT) and heat stress (HS) temperatures and

subcellular fractions pelleted during differential centrifugation were characterized for

aldolase and acid phosphatase enzymatic activities (Figs. 3-4 and 3-5). Relative to CT,

HS treatment significantly increased aldolase activity distributed in pelletable fractions

isolated from E36 cells (Fig. 3-4a). During 8.5 hour incubations that were used,

partitioning of aldolase to pelletable compartments had to be faster than loss. This is

consistent with accumulation of nascent autophagic vacuoles peaking by 6 hours after

autophagic induction (Lawrence and Brown, 1992). Chloroquine (+CHQ) caused a

more significant increase in pelletable aldolase activity. The effect of chloroquine

suggests that lysosomal degradation contributes to aldolase A flux out of pelletable

organelles consistent with an autophagic mechanism. In support of this, accumulation

caused by heat stress and chloroquine (Fig. 3-5, HS+CHQ) corresponds to a 1.50.3%/h

(mean SD, n = 3) increase in the sequestration rate for aldolase A which was similar in

magnitude to induced autophagic degradation for total long-lived proteins (data

presented in a later section). The results support endogenous aldolase A of E36 cells

undergoing autophagic delivery to lysosomes during heat stress.











Aldolase Accumulation
3.0
S,*LP cHP
(0 _^
N 62.5
t..II
0.
S"'2.0 1**

u 1.5
'o


1.0

50.



0.0 1 ; I ;
E36 E36 E36 ts20 ts20
CT HS HS+CHQ CT HS
Cell Line and Treatnment
Figure 3-5: Ubiquitination Mediates Lysosomal Accumulation of Aldolase A During
Heat Stress. Using enzyme activities collected for Figure 3-4, aldolase was divided by
acid phosphatase to indicate relative aldolase associated with organelles (mean + SEM, n
= 3); values were normalized to the 100,000 x g pellet (HP) of control temperature (CT)
to reflect aldolase accumulation relative to unstressed conditions. Labels are as in Figure
3-4. Student's t-test: **, p <0.03; ***, p <0.009.



Heat stress (HS) by itself failed to affect the distribution acid phosphatase activity

in subcellular fractions (Fig. 3-4b). This indicated that increase of aldolase A in pellets

was not due to redistribution of lysosomal organelles and supported the idea that the

aldolase A was undergoing enhanced accumulation. Chloroquine treatment (+CHQ) had

no effect on total pelletable acid phosphatase (Sum) but caused a redistribution of acid

phosphatase from high-speed pellets (HP) to low-speed pellets (LP). It is known that










chloroquine treatment causes lysosomes to swell (Glaumann, et al., 1986). As a weak

base, chloroquine accumulates in organelles proportional to their acidity, and mature

lysosomes are the most acidic organelles. The results here indicate that some lysosomes

became large enough to pellet at lower centrifugation speeds.

A basic result of subcellular fractionation is that different pelleted fractions have

different contents of organelles (Rickwood, 1992). To demonstrate that aldolase A

accumulates in a subpopulation of organelles (presumably lysosomes), aldolase activity

was normalized to acid phosphatase activity and calculated the accumulation of aldolase

A in HP and LP relative to lysosomal content (Fig. 3-5). Significant accumulation of

aldolase activity only occurred in HP fractions during heat stress. In the presence of

chloroquine (+CHQ), there was a greater than two-fold accumulation of aldolase activity

in HP fractions but not LP fractions. The results suggest that aldolase A containing

organelles were preferentially isolated in HP even during CHQ treatment, and are

consistent with heat stress causing accumulation of aldolase A in a subpopulation of

lysosomes.

A previous study has shown that the fractional volume of autophagic vacuoles

and lysosomes does not significantly increase in heat stressed E36 cells (Lenk, et al.,

1992). Together, the data indicate that heat stress increases the flux of aldolase A into

autophagic vacuoles during heat stress. Unlike wildtype E36 cells, heat stress-induced

accumulation of aldolase activity with pelletable organelles failed to occur for mutant

ts20 cells (Figs. 3-4 and 3-5). Since heat stress inhibits ubiquitination in ts20 cells, this









75
suggested that aldolase A accumulation in organelles requires ubiquitination. The data

support a role for ubiquitination in heat-stress induced sequestration of aldolase A.

Using electron microscopic morphometry, a previous study shows that in heat stressed

ts20 cells conversion of autophagic lysosomes into residual bodies is specifically

inhibited, resulting in a 6-fold accumulation of lysosomal volume (Lenk, et al., 1992).

The subcellular fractionation results here indicate that earlier events in autophagic

degradation (aldolase A sequestration) might also involve ubiquitination. In conclusion,

the endogenous aldolase A of E36 cells appears to require ubiquitination for heat-stress

induced delivery to lysosomes.

Heat Stress-Induced Lysosomal Proteolysis of Aldolase B Requires Ubiquitination

Earlier in this chapter, ubiquitinated aldolase B in liver was shown to contribute

to ubiquitin conjugates that are enriched in autophagic organelles during starvation-

induced autophagy. Ubiquitination was required for heat stress-induced delivery of

endogenous aldolase A to lysosomes ofE36 cells, suggesting that aldolase A was

degraded via ubiquitin-mediated autophagy. To examine whether aldolase B undergoes

ubiquitin-mediated autophagy like aldolase A, subcellular fractionation studies in the last

section were repeated with E36 and ts20 cells expressing epitope-tagged aldolase B

(RABM).

E36 and ts20 cells were transfected and selected for permanent expression of rat

aldolase B with the 9E10 myc epitope on its carboxyl terminus (RABM). The 9E10

epitope allowed efficient immunoprecipitation needed for degradation assays and











a.







52


;1~
I,


~.'
I.


Ph .


b.







0
(N1


I nS

'S




0


Figure 3-6: Transient Expression of RABM. a) E36 cells and b) ts20 cells transiently
expressing rat aldolase B with a carboxyl terminal myc tag (RABM) were processed for
immunofluorescence microscopy (Materials and Methods) and labeled with antibody
against aldolase B (ALDB) or the myc epitope (9E 10). Phase contrast (Ph) images for
corresponding fields are shown below each immunofluorescent micrograph. Scale bar =
50 4M.


9E10


4











unambiguous identification of the exogenous aldolase B, RABM. In Figure 3-6,

transient expression of RABM was easily detected in a small fraction of cells with

antibody to either the myc epitope (9E 10) or to native aldolase B (ALDB). Many

unlabeled cells indicated that E36 and ts20 cells do not express endogenous aldolase B.

Cell lines were isolated and screened for permanent RABM expression, and the highest

expressing lines for E36 and ts20 cells were designated E36AB and ts20AB, respectively

(Figs. 3-7 and 3-8).

After clonal selection all cells in a microscopic field were labeled for RABM in

permanent cell lines. Though most cells were brightly labeled, some were only dimly

labeled. Such labeling remained constant after multiple culture passages and for different

cell lines, suggesting that the variability was a trivial artifact of the immunofluorescence

protocol. According to immunofluorescence and western blot assays, different cell lines

had characteristic RABM levels that were maintained after multiple passages (data not

shown). Control experiments performed with cell lines expressing 5 to 10 fold

differences in RABM levels gave similar results. To facilitate detection, the highest

expressing lines (E36AB and ts20AB) were used extensively, and data are reported for

these lines. Immunofluorescent morphology indicated that RABM predominated in the

cytosol as shown by the presence of negatively labeled nuclei and vacuoles, providing

evidence that the recombinant protein demonstrated normal localization (Figs. 3-6, 3-7,

and 3-8).










E36


E36AB


W.. p '.


Figure 3-7: Permanent Expression of RABM in E36 Cells. Transiently transfected E36
cells were selected and screened for permanent RABM expression; the highest
expressing cell line (E36AB) and untransfected cells (E36) were processed for
immunofluorescent detection as described in Materials and Methods with anti-9E10 myc
epitope (9E10, upper panels); phase contrast for corresponding fields are shown (Phase,
lower panels). Scale bar =50 pM.


i'i. it










ts20


ts20AB


Figure 3-8: Permanent Expression of RABM in ts20 Cells. Transiently transfected ts20
cells were selected and screened for permanent RABM expression; the highest
expressing cell line (ts20AB) and untransfected cells (ts20) were processed for
immunofluorescent detection as described in Materials and Methods with anti-9E10 myc
epitope (9E 10, upper panels); phase contrast for corresponding fields are shown (Phase,
lower panels). Scale bar = 50 jtM.











E36 E36AB ts20 ts20AB
1 2 3 1 2 3 1 2 1 2 3


QQ
_g







0


Figure 3-9: Biochemical Detection of RABM Expression. Three confluent cultures (1,
2, 3) for each of the indicated cell lines was trypsinized, pelleted, suspended in 2 x
sample buffer, boiled, divided into two aliquots, run on duplicate SDS-PAGE gels,
western blotted, and duplicated blots were stained for aldolase B (upper panel) or the
9E10 myc epitope (lower panel); E36, original Chinese hamster lung cell line; ts20, an
E36-derived ts-mutant in ubiquitination; E36AB; an E36-derived cell line permanently
expressing RABM; ts20AB, a ts20-derived cell line permanently expressing RABM.
Arrow heads mark molecular weights (kD) for a doublet detected with anti-aldolase B.


-4 41.3
""40.0









-"40.0









On western blots of whole cells labeled with anti-aldolase B (Fig. 3-9), a single

faint 40 kD band was detected in untransfected cells (E36 and ts20), consistent with

cross-reactivity to aldolase A (Fig. 3-2b). Lysates of E36 and ts20 cells had aldolase

cleavage activity 35 to 40 fold higher for fructose 1,6-diphosphate than for fructose-1-

phosphate (data not shown). This difference for the two aldolase substrates is

characteristic for aldolase A, identifying this isoform as the prevalent endogenous

aldolase of E36 cells; note, aldolase B was not detected by immunofluorescence (Fig. 3-

6).

For RABM-expressing cells (E36AB and ts20AB), a closely spaced doublet of

bands was labeled with aldolase B antisera on western blots of whole cells (Fig. 3-9).

One band occurred at 40 kD coincident with endogenous aldolase A of untransfected

cells (E36 and ts20). A second strongly labeled band occurred at SDS-PAGE mobility

corresponding to 41.3 0.1 kD (mean SEM, n = 6). On duplicate blots labeled for the

C-terminal myc epitope of RABM (9E 10), long luminographic exposure times only

showed the 41.3 kD band (Fig.3-9). 41.3 kD matched the predicted molecular weight of

RABM, confirming that E36AB and ts20AB cells express full-length RABM.

If differences between immunoreactivities for hamster aldolase A (E36 cell

endogenous) and rat aldolase B (RABM) are similar to differences between titered

immunoreactivities (Fig. 3-2b) for purified rabbit aldolase A (Sigma) and purified human

aldolase B (Fig. 3-1), then immunoreactivities can be used to estimate RABM expression

relative to endogenous aldolase A. Endogenous aldolase A immunoreactivity was below








82
the linear range of western blot ECL assays in sample sizes subsaturating for aldolase B

detection. Immunodetection in the low range variably underestimated aldolase A by 3 to

>5 fold (data not shown), but allowed an upper limit for relative RABM expression to be

estimated. Estimates varied in the range of 0.2-2.4 RABM per endogenous aldolase A.

Though not precise, these overestimates indicated that RABM levels in permanent lines

were equal or less than the endogenous.

To determine whether RABM undergoes heat stress-induced autophagy, cultures

ofE36AB and ts20AB were incubated at control (CT) and heat stress (HS) temperatures

and fractionated as done for untransfected cells in the previous section titled, "Heat

Stress-Induced Autophagy of Aldolase A Requires Ubiquitination." Subcellular

distributions for activities of aldolase A and acid phosphatase were indistinguishable

between parental and RABM-expressing cell lines. The enzymatic activity for aldolase A

is -10 fold more than aldolase B (Penhoet and Rutter, 1975). Given this, aldolase B

enzymatic activity was too low to detect above endogenous aldolase expression in E36

cells. To overcome this problem, aldolase B immunoreactivity was followed on western

blots of subcellular fractions (Fig. 3-10).

Figure 3-1Oa compares the distribution of immunoreactivities for RABM

(Aldolase B) and an integral membrane protein of lysosomes (LAMP2b) in subcellular

fractions isolated from E36AB cells incubated at control temperatures (CT). Details of

the subcellular fractionation are described in Materials and Methods. When cell sheets

were scraped from culture dishes and pelleted, some soluble proteins leaked out of the

cells, as indicated by the presence of RABM (Aldolase B) in the supernatant (Sc, Fig. 3-















Figure 3-10: RABM Associated With Lysosomes Undergoes Ubiquitin-Mediated
Proteolysis. Confluent E36AB and ts20AB cell cultures were incubated for 8.5 total
hours at indicated conditions and then fractionated as described in Materials and
Methods; samples of fractions were separated by SDS-PAGE, western blotted and
labeled for distribution of lysosomal membranes on higher molecular weight half of blots
(upper panels) and RABM on lower half of blots (lower panels) using antibodies to an
integral membrane protein of lysosomes (LAMP2b) or to native aldolase B (Aldolase B);
a) E36AB cells incubated in CT (see below) conditions were used to show distributions
for lysosomal membranes and RABM which were comparable for all treatments within
the variability of ECL detection (Amersham), 1% total culture equivalent of fractions
was loaded per lane except HP (lane 6) which used 10%: Sc, supernatant from cells
scraped then pelleted at 1000 x g; Ho, homogenate of lysed cell pellet; LP & Lsu, pellet
& supernatant from 1000 x g of Ho; HP & Hsu, pellet & supernatant from 100,000 x g
of Lsu; b) HP fractions isolated from cells incubated under indicated conditions were
loaded for equal acid phosphatase activity to show relative content of full-length RABM
(Aldolase B, 41.3), proteolyzed RABM (Aldolase B, 40.0), and lysosomal membranes
(LAMP2b): Co, control temperature, in DMEM + 10% FBS (normal culturing
conditions); CT, control temperature in MEM medium (experimental control); HS, heat
stress in MEM medium; +CHQ, medium supplemented with 80 PM chloroquine.



Simplified Diagram of Fractions*:

Cell Sheet --> scrape & 1000 x g --> sup = Sc

pel, homogenize = Ho

1000 x g -+ sup = Lsu -> 100,000 x g -*> sup=
Hsu
4-pel = LP pel = HP
pel =LP pel =HP


*sup, supernatants; pel, pellets










a. E36AB CT
Sc Ho LP Lsu Hsu HP

tNI

135
1 2 3 4 5 6


-o- ~ w


b. HP Fractions
E36AB ts20AB


HS+
Co CT HS CHQ


Co CT HS


NOW


1 2 3 4 5 6 7


v --.41.3
-w 40.0


-.41.3
" 40.0









85
10a, lane 1). Membrane-bound organelles were retained in cells as indicated by the lack

of LAMP2b label in Sc.

The cell pellet was lysed, homogenized (Ho, Fig. 3-10a, lane 2), and used to

produce 1000 x g (low speed) pellet (LP) and supernatant (Lsu). Low speed pellets

generally contain nuclei, large cell fragments, and unbroken cells (Rickwood, 1992). LP

contained no detectable RABM, indicating that most cells were broken enough to lose

soluble proteins to Lsu (Fig. 3-10, lanes 3 and 4). Supporting this, LP contained about

15% of the aldolase A activity contained in Lsu (data not shown). The fact that some

aldolase A (Fig. 3-4a) but no RABM was detected in LP is probably due to the greater

affinity of aldolase A for pelletable cell components compared to aldolase B (Kusakabe,

et al., 1997). LAMP2b was approximately equally distributed in LP and Lsu, indicating

that about half the organelles (at least lysosomes) cofractionated with large cell

fragments or nuclei. In agreement with this, acid phosphatase activity was similarly

distributed between LP and Lsu (data not shown).

The Lsu was then used to produce 100,000 x g (high speed) pellet (HP) and

supernatant (Hsu). Such high speed centrifugations pellet all membrane-bound

organelles and leave soluble cytosolic (and leaked organellar) components in the

supernatant (Rickwood, 1992). Consistent with this, all the detectable lysosomal

membranes (Fig. 3-10a, LAMP2b) in Lsu (lane 4) were pelleted out of Hsu (lane 5). To

make RABM (Aldolase B) labeling in HP (Figure 3-10Oa, lane 6) comparable to that

loaded in lanes containing cytosol, 10 fold more HP equivalent was loaded.









86
On western blots of directly harvested whole cells (E36AB and ts20AB), aldolase

B immunoreactivity (RABM) primarily occurred at 41.3 kD (Fig. 3-9). In subcellular

fractions, a large proportion (>40%) of aldolase B immunoreactivity occurred at 40 kD

(Fig. 3-10a). This indicated that 41.3 kD RABM was proteolyzed to -40 kD size during

fractionation. Like RABM, the LAMP2b-reactive protein was also proteolyzed as

indicated by the presence of a smear below its band on western blots (Fig. 3-lOa,

especially visible in 10X loaded HP, lane 6). EDTA and storage on ice was used to

reduce protein degradation in subcellular fractionations but was insufficient to prevent

this presumably artifactual proteolysis. However, this result suggested that processing of

RABM from 41.3 kD to 40 kD could be used as an indicator of proteolysis.

Consistent for all treatments and fractions containing RABM (except HP), the

artifactual proteolysis was limited to processing 40-60% of the RABM (Fig. 3-10a, lanes

1, 2, 4, and 5). Only in HP fractions, processing of 41.3 kD RABM to 40 kD was

complete or nearly complete, such that aldolase B immunoreactivity collapsed from a

doublet to a single band at 40 kD (Fig. 3-1Oa, lane 6; Fig. 3-lOb, lanes 1, 2, 3, 5, and 6).

However, if lysosomal degradation was inhibited by chloroquine (E36AB, HS+CHQ) or

autophagic degradation blocked by the non-permissive ubiquitination of heat stressed

ts20 cells (ts20AB, HS) then the complete processing of RABM in HP fractions was

blocked, as indicated by the persistence of 41.3 kD RABM in a doublet (Fig. 3-lOb,

lanes 4 and 7). These data indicated that RABM proteolysis occurred in lysosomes

(CHQ-sensitive) and required ubiquitination (blocked in HS ts20AB). Whether greater










proteolysis of RABM seen in HP occurred in cells or during fractionation was not

determined. With the data from the last section, the results suggest that RABM

(aldolase B), like endogenous aldolase A, utilizes lysosomal degradation that requires

ubiquitination in E36 cells.

To demonstrate the presence of ubiquitinated aldolase B in E36 cells expressing

RABM, E36AB and ts20AB cell cultures were incubated at the different conditions used

for subcellular fractionation studies above. Then RABM protein was isolated using

9E10-specific immunoprecipitation and separation on SDS-PAGE (Materials and

Methods). Gels were western blotted and detected with native (N) and denatured (D)

aldolase B antisera. Since antisera for denatured aldolase B were most sensitive and

specific for ubiquitinated aldolase B on western blots (Fig. 3-2a and 3-3), these

antibodies were used to probe for ubiquitinated aldolase B (Fig. 3-11 a, upper panel). A

major stable ubiquitin-aldolase B conjugate occurred at 68 kD consistent with Ub68

found in rat liver (Fig. 1-3 and 3-3) and in Fao hepatoma cells (Fig. 1-4). This confirmed

that E36AB and ts20AB cells contained ubiquitinated aldolase B, including Ub68.

Similar levels of Ub68 were detected in all conditions, including heat stressed ts20 cells

(ts20AB, HS MEM) in which ubiquitination is inhibited. Unchanged ubiquitinated

protein under conditions of inhibited ubiquitination appears contradictory. Though

greatly inhibited, a low level of ubiquitination continues in heat stressed ts20 cells, but

this low level is insufficient to mediate cellular processes (Hischberg and Marcus, 1982;

Kulka, et al., 1988; Gropper, et al., 1991). The results here suggest that low levels of

ubiquitination were sufficient to maintain multiubiquitinated intermediates of aldolase B






















Figure 3-11: Ubiquitinated Aldolase B Occurs in E36 and ts20 Cells Expressing RABM.
Replicate sets of E36AB and ts20AB cultures were treated with indicated media
(DMEM +FBS or MEM) and temperatures (CT or HS), harvested, and
immunoprecipitated with 9E 10 antibody; the immunoprecipitate was pelleted (P) from
the lysate and proteins remaining in the supernatant were precipitated with
trichloroacetic acid (S); P and S samples were boiled in 2 x sample buffer, split in equal
aliquots, and duplicate gels run on SDS-PAGE; a) Western blots to detect RABM
immunoreactivity (Aldolase B) were made from one gel and upper and lower portions of
blots were immunodetected with anti-denatured (upper panel, D) and anti-native (lower
panel, N) aldolase B, respectively; b) the duplicate gel was Coomassie stained, lane
numbers at the top of the Coomassie gel correspond to identical samples in lanes
numbered at the bottom of the blots in (a.); MW, molecular weight markers; molecular
weights (kD) are indicated at right.








E36ABts20AB


CT
DMEM+FBS MEM
P S P S


HS CT
MEM DMEM+FBS MEM
P S P S P S


*f lr '-


HS
MEM
P S


9"'


N q .rA


4 *. 68


I


0o


E


S^W^


-.41.3
-40.0


1 2 3 4 5 6 7 8 9 10 11 12
b. MW 1 2 3 4 5 6 7 8 9 10 11 12 MW


-466.2

455.0
442.7
440.0


4 31.0


4 21.5


E36AB


Dt









90
(Ub68 probably contains 4 ubiquitins), but were insufficient to mediate the proteolysis of

RABM detected in HP fractions (Fig. 3-10b, lane 7). The occurrence of Ub68 in

different samples from different cell types (Figs. 1-3, 1-4, and 3-11) supports it as a

stable basal intermediate that probably requires more ubiquitination to facilitate

proteolysis. Alternatively, heat stress-induced ubiquitination could operate on the

machinery of stress-induced degradation. Further experiments are needed to distinguish

these alternatives.

As detected with antiserum to native aldolase B (N), immunoprecipitation with

the 9E10 epitope (specific for RABM) effectively pelleted all detectable 41.3 kD RABM

protein and more than half of a dim-labeled 40 kD protein, presumably endogenous

aldolase A (Fig. 3-1 la, lower panel). The 40 kD aldolase A was shown to lack 9E10

immunoreactivity (Fig. 3-9), indicating that RABM and endogenous aldolase A occur as

a complex in E36AB and ts20AB cells. This is consistent with the known tetrameric

structure of all FDP aldolase isozymes, wherein different subunits randomly and stably

oligomerize during synthesis (reviewed in Chapter 1). Ubiquitinated forms of aldolase B

were also removed from lysates by 9E10 immunoprecipitation (Fig. 3-1 la, upper panel),

suggesting that ubiquitinated RABM retained its C-terminal epitope tag or retained

associations with unmodified 9E10-immuno-reactive RABM subunits. If ubiquitinated

RABM is not associated with other aldolase subunits, then this would provide evidence

that ubiquitination might disassemble quaternary structure of aldolase B perhaps as an

early step in the degradative pathway. However, this possibility was not pursued here.










Experimental
Treatment

A. Control]


Association
With
Organelles


Early
Intermediates


Limited
Lysosomal
Proteolysis


Late
Intermediates


Sbasal Aldolase A 40 kD RABM
(trace, 41.3kDRABM)


B. IHeat Stress Aldolase A 40 kD RABM
(trace, 41.3 kD RABM)

C. HeatStress 1 Aldolase A kD_
+chloroquine : 41.3 kD RABM 40 kD RABM



D. Heat Stress basal Aldolase A 40 kD RABM
ts20 mutant {^ _41.3 kDRABM



Figure 3-12: Summary of Association and Limited Proteolysis of RABM and
Endogenous Aldolase A in Pelleted Organelles of E36 Cells. Each pathway corresponds
to experimental treatment described in boxes at left and correspond to the following
abbreviations used above: A) E36AB or ts20AB, CT; B) E36AB, HS; C) E36AB, HS
+ CHQ; D) ts20AB, HS. Since aldolase A was detected by enzymatic activity and is
inactivated by limited proteolysis, no late intermediates of Aldolase A are shown. Since
RABM detected on western blots shifts from 41.3 kD to 40 kD forms by limited
proteolysis, these forms are listed for early and late intermediates, respectively. Weight
of white vertical arrows indicate relative increases in detected levels of aldolase A and
RABM pelleted with organelles. Weight of black horizontal arrows indicate relative
rates proposed for processes listed in the heading.


Figure 3-12 summarizes the results of subcellular fractionation studies for

endogenous aldolase A and RABM expressed in E36 cells. Aldolase A activity is very

sensitive to proteolytic inactivation and loses 98% of its activity upon limited proteolysis

(Penhoet and Rutter, 1975; Horecker, et al., 1985). Since this made proteolyzed









92
aldolase A undetectable in the background of active aldolase A from E36 cells, aldolase

A activity was used to demonstrate association with organelles but not for detecting

proteolyzed intermediates of degradation. RABM was detected by western blotting with

antiserum to native aldolase B, allowing detection of limited proteolysis (41.3 kD --+ 40

kD) products referred to here as "late intermediates" (Fig. 3-12, last column). Smaller

molecular weight intermediates of proteolysis were not detected, because they are not

recognized by anti-native aldolase B and probably are more rapidly degraded than 40 kD

aldolase B (Reznick, et al., 1985; Horecker, et al., 1985).

Under control conditions (Fig. 3-12, A), basal levels of aldolase A, 40 kD

RABM, and a trace of 41.3 kD RABM were detected in pelletable organelles. Heat

stress (Fig. 3-12, B) caused a partial increase in aldolase A activity with little apparent

change in RABM forms. However, a partial increase in trace levels of 41.3 kD RABM

were likely to be missed, because they were below the threshold for optimal Enhanced

Chemiluminescent detection (Amersham). Consistent with reaching the threshold for

detection, chloroquine inhibition of limited proteolysis caused a sudden signal increase in

41.3 kD RABM (Fig. 3-12, C). Chloroquine also caused an even more aldolase A to

accumulate. The results indicated that lysosomal proteolysis mediates loss of aldolase A

and aldolase B (RABM) associated with organelles, demonstrating sequestration of these

proteins into lysosomes.

When ubiquitination was inhibited by the ts20 mutation (Fig. 3-12, D) different

results were obtained for aldolase A and aldolase B (RABM). Consistent with




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